Animals, castration, and diets

All experimental procedures involving animals were approved by the Chonnam National University (CNU) Institutional Animal Use and Care Committee (permission number CNU IACUC-YB-R-2010-13). All animal management procedures followed the standard operating protocols of CNU. Male Sprague–Dawley rats were purchased from Orient Bio (Gyeonggi-Do, Republic of Korea) and were maintained in a temperature (22 ± 1 °C)- and humidity (45–65%)-controlled room under a 12/12 h light/dark cycle with ad libitum access to food and water.

Rats were castrated at 6 weeks of age, at which time their average body weight was 213 g. All surgical instruments were pre-sterilized by acceptable methods, including steam sterilization. Male rats were anesthetized by intramuscular injection of Zoletil™ 50 (5 mg/kg bodyweight, Virbac, France) and placed on a heating pad. The scrotum of the rats was shaved and cleaned using 70% ethanol. A small midline incision (1 cm) was made through the skin of the scrotum, and the testes were located, gently squeezed out, and removed. Next, the vas deferens, fat, and blood vessels were restored to their original position in the scrotal sac, and the vas deferens and blood vessels were ligated using silk sutures (Ailee, Seoul, Korea). The muscle layer and skin were closed with sutures, after which the rats were returned to their cages and their recovery monitored. The sham surgery control rats underwent an identical procedure as castrated rats except no removal of the testes.

After acclimatization for 1 week following castration with ND feeding, the rats were divided into the following four groups (8 per group): sham-operated control rats fed an ND or HFD and castrated rats fed an ND or HFD. The normal AIN93-G diet (D10012G) and HFD (D12451) were purchased from Research Diets, Inc. (New Brunswick, NJ, USA); their compositions are shown in Supporting Tables S1-S3. Briefly, in the HFD versus ND, 45% versus 16% of energy was from fat, 35% versus 64% from carbohydrates, and 20% versus 20% from protein, respectively. The food intake of the rats was recorded daily at the same time of day, and their body weight was measured weekly.