Two to one intermittentt fasting regimen comprise of one day of fasting followed by two days of feeding makes it possible to study the effects of dietary interventions involving periodic energy restriction rather than the effects of calorie intake. Unlike alternate day fasting, which results in under feeding two to one intermittent fasting provides mice with enough time to fully compensate for the energy loss and body weight reduction, which happen during fasting. This allows scientists to examine the effects of periodic fasting independent of calorie intake.
Two to one intermittent fasting promotes a number of benefits against diet in used in obesity and associated metabolic dysfunction including impaired glucose homeostasis and fatty liver disease. This protocol can be easily adjusted to a five to two diet regimen comprised of five days of feeding and two days of fasting or time restricted feeding where access to food is limited to eight hours per day. If one uses a mouse model, that shows alternative feeding behavior, such as hyperphagia, ad libitum feeding may not be a proper control condition.
In this case, a pair feeding rule will be required to examine and compare the effects of intermittent fasting. Start by preparing a normal diet or a high fat diet for lean and diet induced obesity mouse models. Measure the baseline body weight and composition of each seven week old male C57BL/6J mouse using a scale and EchomRI, respectively.
Based on the weight and body composition results randomly and equally divide the mice into two groups:ad libitum or AL, an intermittent fasting or IF groups. Then, place two or three mice per cage ensuring that the mice have free access to drinking water. Allow the mice one week to acclimate to the new cage environment prior to starting the IF regimen.
When ready to start the fasting period, move the mice to a clean cage with fresh bedding at 12:00 p.m. Provide a weighted amount of food to the AL group. No food to the IF group.
After 24 hours, weigh the mice in both groups as well as the leftover food in the AL cages. At 12:00 p. m, provide a weighted amount of food to both AL and IF groups.
Once the feeding period is over measure the weight of the mice and leftover food. Repeat the cycle for the duration of the study. Set up a pair feeding or PF controlled group for the duration of the experiment in order to ensure a calorie independent comparison to IF.Measure the amount of food consumed by the IF group.
Divide it evenly into three portions and provide one portion daily to the PF control. When working with animals with altered eating behaviors, such as hyperphagia, it is critical to provide an equal amount of food daily to the pair-fed control group in order to prevent mice from eating the food all at once. To analyze body composition of the mice turn on the body composition analyzer, and leave the machine on for two to three hours to warm up.
Prior to using it, run a system test to ensure measurement accuracy. And if necessary, calibrate the system with canola oil. Measure the body weight of the each mouse.
Then, place it in the small animal cylindric holder. Insert a delimiter to constrain physical movement of the mouse during measurement. Place the holder into the body composition analyzer.
Run the scanning program, which should take 90 to 120 seconds. Then, remove the holder from the equipment. Place the mouse back in the cage.
Conduct a glucose and insulin tolerance test to examine the effects of intermittent fasting on glucose homeostasis. After overnight or six hour fasting, perform an intraperitoneal injection of glucose or insulin on each mouse. Then, measure the blood glucose at 0, 5, 15, 30, 60, and 120 minutes post injection using a glucometer.
To investigate the effects of intermittent fasting on energy metabolism, perform indirect calorimetry over a single cycle of IF.After the mice have acclimatize to the system, fast them by removing food and crumbs from the hopper and the bottom of the cage. Re-introduce food after 24 hours for the two day refeeding period. At the end of the IF cycle, bring the mice back to their original cage and collect data from the program.
The mice that fasted for 24 hours experienced up to 10%reduction in body weight, which was fully recovered after two days of refeeding. When comparing energy intake between one to one and two to one intermittent fasting, it was found that one day of refeeding period was not sufficient to compensate for the caloric lost that occurred while fasting. Compared to AL, IF treatment led to lower body weight increase in wild-type mice fed with normal chow or high fat diet without significant differences in food intake.
Body composition analysis revealed that IF reduced fat mass without changing lean mass in wild-type mice. Experiments with the pair-feeding control confirmed that the decreased body weight gain by IF was not due to altered energy intake in wild-type mice. However, in genetically obese, ob/ob, mice pair-fed control group was indistinguishable from the IF group in body weight and body composition.
Glucose and insulin tolerance tests showed that high fat diet IF mice exhibited significant improvement in glucose homeostasis compared to high fat diet AL and high fat diet pair-fed mice. In ob/ob mice, the IF group exhibited significantly improved glucose handling with smaller glucose excursions compared to the pair-fed control group with unaffected insulin sensitivity. Another metabolic effect of IF noted in wild-type mice was higher oxygen consumption indicative of energy expenditure specifically during feeding period.
Feeding behavior of mice can be affected by environmental factors including housing density. Therefore, it is important to keep the same number of mice housed per cage between groups and over the course of the study. When employing a new mouse model, it is recommended to examine the feeding behavior before performing an intermittent fasting experiment.
Isocaloric two to one intermittent fasting can be easily applied to other disease models including diabetes, heart disease, atherosclerosis, and neurological diseases.
