Antibiotics cocktail

HUVECs were purchased from Lonza and maintained in Endothelial Cell Growth Medium MV 2 Kit (Takara Bio Inc). U937 cells were purchased from American Tissue Culture Collection and maintained in RPMI-1640 medium including 10% fetal bovine serum supplemented with 2 mmol/l glutamine and an antibiotics cocktail (Nacalai Tesque). We used 8-week-old male C57BL/6, nondiabetic (db/m; BKS.Cg-m+/+Lepr^db/Jcl) and diabetic (db/db; BKS.Cg-+Lepr^db/+Lepr^db/Jcl) mice, which were purchased from CLEA Japan Inc. DPPIII (10 μg/g body weight) or PBS was intravenously injected into mice through the tail vein three times weekly for 8 weeks as previously described (30 (link)). Mice were housed in specific pathogen-free conditions in the Research Center for Animal Life Science of Shiga University of Medical Science. The animal experiments conducted in this study were approved by Shiga University of Medical Science Animal Care and Use Committee and were performed in accordance with relevant guidelines and regulations, including Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines.

FMT was performed as previously described [20 (link)]. For the bacterial transfer experiments, donor mice received PEG or saline orally for two weeks, and 4–5 fecal pellets (~100 mg) were collected fresh for daily FMT. The collected feces were suspended in 1 mL of phosphate-buffered saline, followed by centrifugation at 2000× g for 10 s. To minimize the changes in microbial composition of the supernatant, the 100 μL aliquots of the supernatant containing fecal bacteria were orally administered to recipient mice immediately after centrifugation. Recipient mice were fed a HFD for four weeks, treated with a mixture of four antibiotics for four days and subsequently rested for three days prior to transfer. For antibiotic treatment, an antibiotics cocktail containing ampicillin, neomycin, metronidazole, and vancomycin (Nacalai Tesque) was diluted in sterilized water to final concentrations of 5, 5, 5, and 2.5 mg/mL, respectively. The antibiotics cocktail (200 mL) was orally administered for four days. Oral antibiotic administration via ad libitum administration via drinking water was avoided to minimize individual differences in body weight change and antibiotic doses [26 (link)]. After three days of rest following antibiotic administration, FMT from donor mice was performed five times weekly for six weeks.