Restricting the timing of food intake has emerged as a promising intervention to attenuate diet-induced metabolic diseases. This protocol details the construction and use of an efficient system, built in-house, for measuring and manipulating rhythmic food intake in mice. The main advantage of the system is that it's a low-cost and efficient system that can be built using affordable materials, while being user-friendly at the same time.
This system can be used to feed mice on different feeding regimens, such as ad libitum, time-restricted, or arrhythmic schedules, and can incorporate high-fat diet to study their effects on physiology. To begin, procure four pieces of 0.25 inch PVC sheets. Cut and glue the four pieces of PVC to obtain the base.
Next, open the timer to remove the plug, and make a connection using a standard extension or electric cord. Place the timer on the PVC base to align it with the holes drilled into it, and fix using 1.5 inch screws. Drill four holes on top of the timer, and fix 0.75 inch screws to hold the eight-compartment food container.
Use a mouse cage having a width greater than 4.5 inches, and cut a 4.5 inch hole at the bottom of the cage, using a hole saw. Then make a food dispenser using a four-inch, eight-compartment jewelry organizer. Cut the rim of the container, such that it fits nicely into the pipe.
Use a four-inch PVC pipe cap and cut a hole corresponding to the size of a single dispenser compartment to create an opening that exposes only one of the eight compartments at a time. Once all the cages are set, the final setup is ready. To facilitate easier transport, take three pieces of 0.25 inch PVC pipe, drill a 0.625 inch hole in the center of two pieces and glue them together.
Then pass a 0.625 inch PVC pipe through them. Food cups can then be stacked on this assembly. Assess the timers before introducing the mice by plugging the setup into power outlets.
Place a piece of nestlet in one compartment at a recorded time, and monitor the position of the nestlet six hours later to ensure that the timer turns promptly. Transfer the mouse to the experimental room and acclimate them to the light-dark cycle set, as described in the manuscript. Record mouse weight before individually housing them in the feeder cages, and ensure that the mouse has ad libitum access to water, bedding, and nestlets.
Add 1.5 grams of food to all eight compartments of the feeder cup and place the feeder cup on the timer. Then put the lid on the feeder cup, such that only one compartment is exposed. Four compartments represent daytime points, and the other four represent nighttime points.
Change the food every day at the same time, and count the number of pellets remaining in each compartment to calculate the amount of food consumed. Monitor the feeding profile for a week to get a baseline feeding profile of mice fed ad libitum and for the night-restricted diet. After three to seven days of ad libitum diet, put the mouse on a transition diet by gradually reducing the number of pellets in the day compartments.
Monitor the food intake for two weeks after the mouse has adapted to the night-restricted regimen. During this period, adjust the amount of food given to each mouse to better suit its total food consumption. Weigh the mouse at the end of the two weeks to monitor any weight change due to the feeding regimen.
After a week of ad libitum diet, calculate the average daily food consumption and the amount of food to be provided in each compartment, as described in the manuscript. Achieve arrhythmic feeding by ensuring that mice get an equal amount of food in all eight compartments throughout the day. Then put the mice on a transition diet by gradually reducing the amount of food given per compartment, over three to five days, to abolish any rhythm of food intake.
During the arrhythmic diet, adjust the food daily to ensure that mice leave only a few pellets behind and get the right amount of food. Make adjustments as described in the manuscript. To monitor any weight change due to the feeding regimen, weigh the mouse at the end of the two weeks.
The feeding profile indicates that wild-type mice fed normal chow ad libitum eat about 75%of their food during the night. Mice fed high-fat diet ad libitum ate more food on the first two days of exposure, likely because of the novelty of high-fat diet. The daily average food intake every three hours.
The average and the percentage of food intake during the day and night for mice fed with normal chow or high-fat diet was comparable. Male mice showed significant weight gain after one week of ad libitum normal chow and high-fat diet. Mice who transitioned to a night-restricted diet ate their total daily calories only at night, without significantly decreasing calorie intake for the first three to five weeks.
Mice who transitioned to an arrhythmic diet consumed their total daily calories in equal quantities across the day, dramatically dampening their daily food intake rhythm. Mice showed weight gain after the night-restricted and arrhythmic feeding regime. The most important thing to remember when using this feeding system is to ensure that the timers function appropriately throughout the experiment.
Using this feeding system, researchers can customize food delivery according to their requirements, which could eventually help them answer questions about the effects of varied meal timings on health and disease.
