Animals

Three-week-old male mice (n = 65) were purchased from Charles River CD1-IGS (Wilmington, MA, USA). Animals were housed in AAA-LAC accredited facilities under standard conditions (12-hour light/dark cycle, food and water available ad libitum). All experiments were conducted under protocol M005599 approved by the University of Wisconsin Institutional Animal Care and Use Committee. After a 10-day acclimatization period, animals were divided into experimental groups (n = 10–12). Animals’ wellbeing was monitored daily and body weight was recorded three times per week. At the end of experiments, animals were euthanized by the overdose of isoflurane followed by collection of blood via cardiac puncture and collection of spleen tissue. Blood was allowed to clot for 30–40 min at room temperature followed by centrifugation for 10 minutes. Serum was immediately transferred to cryovials and frozen at -80°C until further use. Spleen tissues were frozen immediately on dry ice and stored at -80°C until further use. All animals were sacrificed between 11am and 1pm to minimize differences in serum leptin and adiponectin due to diurnal rhythm [33–35].