Gut microbial metabolites reveal diet-dependent metabolic changes induced by nicotine administration

Male C57BL/6J wild-type mice were housed in a conventional animal room at 23.0°C with a 12-hour light-dark cycle. At 7 weeks of age, the male mice with comparable average bodyweight were divided into two groups: the normal diet (ND) group and the high-fat diet (HFD) group and received twice daily injections of nicotine (0.75 mg/kg bodyweight, intraperitoneal administration) or saline for 4 weeks. For oral nicotine delivery model experiments, 7-week-old male mice were received a nicotine solution (200 mg/mL in 2% saccharin vehicle) in drinking water during the 4-week period of HFD exposure. Feces were collected at 11 weeks of age. Fecal DNA was extracted from frozen samples using the FastDNA® SPIN Kit for Feces. The V4 region of the 16S rRNA gene was amplified using dual-indexed primers. Sequencing of the amplicons was carried out on an Illumina MiSeq instrument with a MiSeq Reagent Kit V3, following established protocols.