C57bl 6n

Each of Macf1 and Ehd1 mutant mouse line was maintained breeding with C57BL6/N (CLEA Japan, Inc.). The mice for behavioral tests were obtained by in vitro fertilization using sperms of the heterozygous mutant mice and eggs derived from C57BL6/N (CLEA Japan, Inc.). Genotyping of all mutant mice was performed using genomic DNA derived from tail tips. Tail biopsies were conducted on postnatal day 14, and each tail tip was incubated in 100 μl of lysis buffer (25 mm NaOH, 0.2 mm EDTA) at 95°C for 30 min. An equal volume of 40 mm TRIZMA hydrochloride was added to the lysate after incubation and the mixture was vortexed briefly. Afterward, 1 μl of the mixture was diluted with water 2.5-fold and were amplified by the PCR conditions shown in Supplementary Material, Table S2C. The PCR products were sequenced with BigDye Terminator V3.1 and ABI 3730xl sequencer (Life Technologies) using sequencing primers (Supplementary Material, Table S2D).

Specific pathogen-free (SPF) 7- or 50-week-old C57BL/6N and KK-Ay/TaJcl male mice were obtained from CLEA Japan Inc. (Tokyo, Japan). The mice were separately kept in cages in specific pathogen-free conditions at 23 ± 1 °C, with a 12 h light:12 h darkness schedule. The mice were housed with unlimited access to drinking water and a pelleted basal diet. On the final day of the study, the body weights were measured. Blood glucose levels were measured from the tail vein of each mouse. We defined diabetic as more than a 300 mg/dL blood glucose level. In order to eliminate factors affecting the experiment, each animal was kept under constant conditions, such as free access to the same food, minimization of stress load, and freedom of movement within the cage. This work was conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of Suzuka University of Medical Science (approval number: 34). All dissections were performed under pentobarbital anesthesia. All efforts were made to minimize mouse suffering.