Intermittent fasting is an umbrella term for dietary plans that are characterized by time-restricted eating. Nowadays, it has become a highly popular weight loss regime, with “every-other-day-fasting” and “5:2” diets gaining more and more followers.
Intermittent fasting comes in two flavors, as it may or may not be combined with caloric restriction. In the first case, the subject receives less food in well-defined periods of the day. The length of the fasting periods and the reduction of caloric intake can vary significantly in different protocols. However, there are several intermittent fasting protocols that solely rely on a strict feeding schedule, while allowing the total caloric input to be the same as before the diet. In this case, the subject eats proportionally more food during the feeding period, but the feeding period itself is limited in a small part of the day. In the end, total food consumption is constant.
Fasting is not a breakthrough; it has been a common practice for thousands of years and is suggested in numerous religions, where it’s believed to serve purifying and cleansing purposes. But beyond that, can fasting benefit our physiology? Are there potential adverse effects upon certain conditions? Today, science is not based on empirical observations of interesting facts. Modern standards of scientific work require the assessment of any given question under strictly controlled and highly repeatable conditions. Animal models, and especially rodents, are invaluable tools in this process, and an increasing body of evidence supports that fasting can be beneficial for human physiology, and ameliorate the symptoms and quality of life in several diseases. While obesity is the obvious one, the list also includes neurodegenerative diseases, cancer, diabetes, cardiovascular diseases, and sleep disorders. In the present article, we will discuss in detail how intermittent fasting can be modeled in rodents and compare different protocols that are widely used in the field of intermittent fasting research.
How to model intermittent fasting
Intermittent fasting is not just about cutting down the food supply. There are specific protocols to be followed, which guarantee an effective outcome while at the same time preventing the devastating adverse effects of malnutrition. In rodents, numerous approaches have been implemented by different research groups, in order to study intermittent fasting. In this section, we will briefly describe the most widely used regimens, emphasizing on the feeding time, quantity of food and the total duration of the protocol. These protocols include the alternate day fasting, the periodic fasting and the time-restricted feeding.
- For the alternate day fasting protocol, mice are subjected to repeated cycles of 24 hours fasting followed by 24 hours of unlimited food access. This is a harsh protocol that cannot be followed for a long period of time, as upon prolonged application it has detrimental effects on mice body weight. A lighter version of the alternate day fasting protocol suggests a 70% energy restriction every other day, rather than complete abstinence from food and energy drinks.
- Periodic fasting protocols / Fasting mimicking diets are more kind approaches, as they allow for an increased ratio of feeding to fasting periods. Common practices involve 2:1, 5:2 and 4:3 feeding to fasting periods that usually last 24 hours each. In all these protocols, mice have unlimited access to food during the feeding periods and are completely restricted from eating during the fasting periods. In the 2:1 protocol, mice have access to food for 48 hours and then are forbidden from eating for 24 hours. In the 5:2 protocol, mice have access to food during 5 out of 7 days of the week and abstain from food during 2 out of the 7 days. Similarly in the 4:3 protocol, mice are feeding for 4 out of 7 days and fasting for the remaining 3 days of each week. An important element of these protocols is that the fasting periods are always arranged in non-consecutive days, and every fasting period is followed by a feeding period, to allow the rodents enough time for recovery from the fasting. According to the literature, these regimens are suitable for long term implementation, which can last up to several weeks or months.
- Last but not least is the time-restricted feeding, an intermittent fasting protocol characterized by an eating pattern in which food intake is restricted to a time window of 8 hours or less every day. This protocol does not necessarily result in the reduction of caloric input, rather, it emphasizes the importance of well-defined feeding periods that are interchanging with long fasting periods on a daily basis. Since the amount of food is not restricted, this protocol can also be followed in long-term experiments.
At this point, it is important to note that most studies in the field of caloric restriction do in fact implement feeding protocols that fall into the category of time-restricted feeding. This originates from the fact that even though in these protocols mice are provided with food every day, their daily food is given in one portion. Under these circumstances, rodents consume it within a period of several hours, thus the remaining 16-20 hours are fasted, in essence following an intermittent fasting protocol. However, there is an alternative approach if one needs to dissociate the effects of time-restricted feeding and caloric restriction. In that case, the experimenter should dilute the provided food with non-digestible material that is commercially available, while allowing the rodent to have free access to food. Non-digestible material consists of fibers, which even though are consumed by the rodent, they cannot be digested and do not provide energy. By diluting the commonly used chow with pellets of non-digestible fibers, the experimenter can ensure that even though mice have unlimited access to food, their energy intake is reduced compared to the control mice, that have access to regular food.
Validation of the fasting
In a number of scientific publications in the field of intermittent fasting, the fasting protocol is not further validated, just described in the Material and Methods’ section and of course, strictly followed. The lack of evidence validating the success of the fasting protocol may be attributed to two reasons. Firstly, several fasting protocols are well-established and the scientific community does not require further proof of their success. Secondly, a fasting protocol is a straightforward approach, and most of the times it elicits a response that is obvious with respect to the experimental question. Thus, by simply comparing the fasted to non-fasted mice, the success of the fasting protocol can be guaranteed.
On the other hand, several studies present actual data that confirm the physiological effect of fasting. In many cases, this also serves as an indication of the sustainability of the protocol, providing evidence that the intervention is not harmful to the rodent physiology. Among the most widely assessed parameters are physiological and biochemical measurement, including body weight, accumulation of body fat and hormone levels.
In the alternate day fasting and periodic fasting protocols, intermittent fasting is combined with the reduction of food consumption, as for a specific number of days per week the rodent is not supplied with food. In this case, the most prominent effect of the intermittent fasting diet is the reduction of body mass and weight loss. Compared to ad-libitum fed animals, fasted animals exhibit a body weight reduction ranging from 5 to 30 %, depending on the specific regimen followed and the rodent strain. When milder protocols are followed, the reduction of body weight might be periodical, as the mice are able to regain the lost weight during the feeding period, so the body weight of rodents is reported on a daily basis. In most cases, in parallel with body mass, researchers record food consumption or total energy intake, in terms of mean gr or Kcal per day. Since the result of intermittent fasting highly depends on the specific protocol, it is advisable for researchers to validate their fasting protocols by evaluating the same parameters as in the original publication.
In all intermittent fasting protocols, independently of whether they require total caloric input reduction, body fat is reduced. This observation is clearer in the context of obesity, where the majority of publications include images of tissue sections with decreased visceral fat, as a phenotypic validation of the success of the fasting protocol.
Glucose and hormone levels
Glucose and insulin levels in the bloodstream are highly regulated by the feeding state of the organism and thus serve as a good indicator of the fasting protocol. Upon fasting, both glucose and insulin levels are decreased, however, the specific time points during which the glucose and hormone levels must be assessed varies among different studies. Furthermore, fasting can reduce insulin resistance, measured by the homeostasis model assessment of insulin resistance (HOMA-IR), which in turn reflects improved hepatic insulin sensitivity. The advantage of assessing biochemical parameters is that they have increased sensitivity and are easily quantified.
Lastly, body temperature is also regulated in response to fasting. Several studies report the reduction of body temperature during the fasting period, compared to feeding period.
The effect of intermittent fasting on rodent behavior
Fasting is reported to up-regulate BDNF (brain-derived neurotrophic factor) levels in the brain. BDNF is a master regulator of neuronal physiology and function and is involved in the neuronal and brain homeostasis. Experimental evidence supports that the fasting-induced increase of BDNF levels is associated with enhanced neurogenesis from neural stem cells, and increased synaptic plasticity.
Apart from physiological measurements, intermittent fasting can also alter the rodent behavior. Several studies have focused on unraveling the fasting-induced changes, as well as their underlying molecular mechanisms. Apart from the role of BDNF, in many cases, the beneficial effects of intermittent fasting are attributed to the down-regulation of the mTOR pathway, which is the sensory hub of nutrient availability in the cell. Upon fasting conditions, the mTOR is suppressed and subsequently, autophagy is induced. Autophagy is a major catabolic pathway in the cell, that degrades proteins, macromolecules, and organelles, to provide energy and building blocks, as well as to ensure quality control of the intracellular constituents. However, the effects of both intermittent fasting and autophagy are not beneficial under all conditions. There are documented cases, such as in hypercholesterolemic mice and mice that suffer a viral infection, that report negative consequences upon fasting. Yet, it seems that in wild type rodents, intermittent fasting has positive results, and improves several aspects of their behavior.
Memory and learning
Fontan-Lozano and colleagues studied the effects of long term intermittent fasting on wild type mice. They employed a battery of behavioral tests to prove that intermittent fasting improves learning and memory, as indicated by enhanced motor coordination in the rotarod test, increased associative learning in the trace eyeblink conditioning test, as well as enhanced short and long term memory validated in variations of object recognition memory test. They further explored the underlying neurobiological mechanisms of intermittent fasting-induced alterations using molecular and electrophysiological studies and found increased long-term potentiation in hippocampal synapses and upregulated levels of the NMDA receptor, all of which are known to serve memory and learning functions in the brain.
These findings were further validated in another study, employing a prolonged intermittent fasting regime in wild type mice. Using the contextual fear conditioning paradigm and the Barnes maze test, the researchers found enhanced memory in fasted mice, as indicated by increased freezing time and reduced time to find the target box respectively.
Behavioral studies in the field of stress indicate that intermittent fasting may indeed ameliorate behavioral aspects of stress. Several publications report increased exploration time in the open arms compartments of the elevated plus maze test, as well as decreased time spent in the peripheral areas of the open field test, both of which indicate a reduced-anxiety phenotype.
In addition, feeding time can have a major influence on circadian rhythms, and intermittent fasting has been shown to regulate aspects of the internal clock. For instance, in time-restricted feeding protocols, rodent activity is increased in a 1 – 2 hours’ time period preceding the time they are fed. This effect may be attributed to the regulation of the hypothalamic suprachiasmatic nucleus function, which constitutes the central circadian control center. Furthermore, studies in Clock deficient mice, which display deregulated circadian rhythms, indicate that intermittent fasting may restore the expression pattern of genes that normally oscillate in the day-night cycle. 
Differential effect of dietary restriction in rodent strains
Intermittent fasting has emerged as an alternative approach to harness the beneficial effects of dietary interventions in order to promote healthspan and lifespan. Apart from specific genetic models, numerous rodent strains as used worldwide to better capture the complexity of human pathology. Currently, there are no studies comparing the effect of intermittent fasting among different strains, however, during the past years several lines of evidence highlight the importance of rodent strains on broader dietary intervention schemes, such as caloric restriction. In a recent study assessing the therapeutic potential of caloric restriction on the age-dependent loss of skeletal muscle mass, researchers showed that the beneficial effects observed in C57Bl/6 mice were abolished in the short-lived DBA/2 mice.
A comprehensive meta-analysis of studies on caloric restriction evaluated the responses of different mice and rat strains upon dietary restriction. Their analysis indicated that the CH3 strain, a model for metabolic syndrome, does not have a consistent beneficial effect upon fasting regarding its median lifespan extension. This effect may be attributed to their increased sensitivity to weight loss, that potentially masks the other positive effects of dietary restriction.
Translational value of preclinical studies on intermittent fasting
Even though intermittent fasting appears to serve outstanding beneficial effects in rodent models of human diseases, several concerns arise regarding its practical applications in human patients. Indeed, testing an intermittent fasting regimen in an animal model is very different from what is practical and safe for humans. Alternate day complete fasting is practically non-applicable in humans, but the reduction of caloric restriction every other day or milder regimens such as 5:2 or 4:3 are commonly adopted by individuals.
A meta-analysis of 14 human-focused studies indicated that intermittent fasting is an effective approach to reduce weight in overweight and obese adults. Interestingly, additional studies report a reduction in depression and improved body image after 8 weeks of following the intermittent fasting diet. However, other studies suggest that intermittent fasting in normal weight adults has adverse effects, as it leads to an increased feeling of hunger, heightened irritability, difficulties in concentrating, increased fatigue, eating-related thoughts and over eating during non-restricted days. Up to date, no studies have been conducted in children and adolescents. Even though all accounted, intermittent fasting seems to be a feasible alternative to caloric restriction, additional studies are necessary to validate its safety under certain conditions and to further support its beneficial effects.
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