Animals.

Six-month-old male Fisher 344 × Brown Norway (F344BN) rats (n = 24) were obtained from the National Institute on Aging Colony at Charles River Laboratories (Wilmington, MA). The number of animals per group was determined by an a priori power calculation that was based on published blood pressure data in rats treated with MTII (27). We determined that a sample size of six animals per group was sufficient to achieve 80% power with 5% α-level. Adult F344BN rats were selected because they display relatively stable body weight under ad libitum access to food (1). Upon arrival, animals were housed under standard conditions, individually with corn cob bedding on a 12:12-h light-dark cycle (lights on at 7 AM). Rats were initially fed a standard rodent chow (18% kcal from fat, no sucrose, 3.1 kcal/g, diet 2018; Harlan Teklad, Madison, WI) and remained on this diet for 7 days to assess baseline light-phase versus dark-phase food consumption. At day 7, all animals were then switched to a novel high-fat diet (60% kcal from fat, 20% protein, 20% carbohydrate, including 6.8% sucrose; 5.24 kcal/g, diet 12492; Research Diet, Brunswick, NJ) for the remainder of the experiment. The objective of this study was to examine the consequences of an obesogenic diet without and with scheduled feeding on obesity and hypertension with a pre (standard diet) and post (obesogenic diet) design; thus, the standard diet was used only before the introduction of the obesogenic diet. In addition to HF content, several components of the obesogenic diet, such as sugar, cholesterol, and phytoestrogens may have also contributed to observed results (39). A transient hyperphagia occurs when rats are introduced a novel high-fat diet (22). Because the role of the melanocortin system in this process is unknown, all rats were allowed a second acclimation period until their daily caloric intake was normalized to the baseline chow diet value.

There is disparity in initial body weight since the National Institute on Aging is the only vendor breeding F344BN, and rats can only be ordered by age, not by weight. Also, response to a high-fat diet is highly variable. Therefore, at day 17, rats were assigned to experimental groups based on body weight to ensure similar baseline value distributions across groups for longitudinal statistical comparisons. Health status, body weight, and food intake were monitored twice daily throughout the experiment. All experimental protocols were approved by the University of Florida’s Animal Care and Use Committee, and in compliance with the “Guide for the Care and Use of Laboratory Animals” guidelines. We attempted to replace animals by other models, but other organisms are not appropriate to evaluate the physiological effect of scheduled feeding or MTII. We refined our techniques and employed a nonsurgical approach to reduce pain in animals. This is a widely used method to measure blood pressure instead of telemetry devices that must be implanted under anesthesia. We also aimed at reducing the number of animals, by conducting an experiment that would use two separate approaches using a single control group. Because the same animals were used as controls for both experiments, all data sets were statistically analyzed together. However, for clarity, data are presented separately as study 1 and study 2.