Hfd 60

All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Science Council of Japan, and they were approved by the Animal Experiment Committee of Gunma University. All experiments were performed in wild-type C57BL/6 J male mice, obtained from CLEA Japan (Tokyo, Japan). Male mice, ages 7–11 weeks, were maintained on a 12-h light/dark cycle at 25 °C and they were given free access to NC and tap water before the experiment. The NC (CE-2) and HFD (HFD32) were purchased from CLEA Japan. Another HFD (HFD60) was purchased from Oriental Yeast Co. (Tokyo, Japan). The HFD32 and HFD60 had different fatty acid compositions (Table 1; information provided by manufacturers). The HFD portions were changed daily to prevent oxidation of fatty acids in the diet. The light/dark cycle was 0600 h/1800 h in the 4-week study and 0800 h/2000 h in the studies that employed the feeding, drinking, and activity monitoring system (FDAMS).

C57BL/6 mice were fed a normal diet (ND) with 5% fat (MF: Oriental Yeast Co, Ltd., Japan), or high fat diet (HFD) containing 60% fat per calorie (HFD-60: Oriental Yeast Co. Ltd. Japan). For obesity experiments, HFD feeding was started at 4 weeks after birth. Adipose tissues and gingival tissues were obtained at 12 weeks after sacrificing the mice.

All animal experiments were approved by the Committee for Care and Use of Laboratory Animals at Kochi University (approval no. J-0100) and followed the guidelines of ARRIVE. All experiments were performed in accordance with the relevant guidelines and regulations. C57BL/6 mice were used in this study. Organs were removed from sacrificed mice and then flash-frozen by liquid nitrogen or fixed in 10% phosphate-buffered formalin for further analysis. For the high fat diet, mice at 5 weeks of age were randomly assigned into two groups (normal diet or high fat diet groups). Mice were fed with standard-chow or high-fat diet (HFD-60, 60% fat content, Oriental Yeast, Tokyo, Japan) for 22–26 weeks.

For the high-fat diet experiment, eight-week-old mice (n = 5) were fed with a control diet (MF diet, Oriental Yeast Co., Tokyo, Japan) or a high fat diet (HFD-60; 60 kcal% fat, 33 kcal% lard and 5.5 kcal% sucrose, Oriental Yeast Co., Ltd.). Body weights were recorded two or three times each week during the experiment. The mice were anesthetized with an intraperitoneal injection of chloral hydrate and then analyzed with a CT scanning machine (Latheta LCT-200, Hitachi Aloka Medical, Tokyo, Japan) to determine the weights of muscle and fat tissues. To estimate the weights of these organs, volume (cm³) was multiplied by density (cm³/g) which is 1.06 or 0.92 for muscle or fat tissue, respectively, according to a manual for the CT machine.

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Hiroshima University Animal Research Committee and AVMA Guidelines on Euthanasia. The protocol described below was approved by the Committee on the Ethics of Animal Experiments of the Hiroshima University (Permit Number: A09-89). All mice were housed in a specific pathogen free facility in 12 hr light-dark cycles with access to water ad libitum.
A total number of 24 5-week-old male SPF C57BL/6J mice (Charles River Japan, Inc., Yokohama, Japan) were randomly divided into two groups, fed either a normal chow diet (CD) or a high fat diet (HFD-60; Oriental Yeast Co., Ltd., Tokyo, Japan). After establishment of the obese mouse model by 12 weeks of HFD feeding, mice were further divided into two subgroups (N = 6 each): Mice in the HFD-Pg group were infected by Pg (as detailed below), while the HFD-NC group served as the negative control without infection. Similarly, the CD group was subdivided and further treated to make CD-NC and CD-Pg groups.

C57BL/6J mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). APP23 mice, which express human APP₇₅₁ cDNA and a Swedish double mutation on the C57BL/6 genetic background^{73 (link)}, were kindly provided by Dr. M. Staufenbiel (Novartis Pharma Ltd; Basel, Switzerland). Obese and diabetic db/db (Lepr^db/db) mice on the C57BL/6 genetic background were purchased from The Jackson Laboratory (Bar Harbor, ME). The mice were housed in a temperature- and light-controlled room (24 °C; 12-h light/dark cycle) and fed on AIN93G (standard diet) or HFD-60 (Oriental Yeast Co., Ltd; Tokyo, Japan). All animal studies were approved by the Gifu University Graduate School of Medicine Animal Care and Use Committee and were performed in accordance with the guidelines for experiments on animals provided by the Ministry of Education, Culture, Sports, Science and Technology of Japan. All animal experiments were performed according to the ARRIVE guidelines.