K.-H.K was supported by the Heart and Stroke Foundation of Canada Grant-in-Aid (G-18-0022213), J. P. Bickell Foundation and the University of Ottawa Heart Institute Start-up fund; H.-K.S. was supported by grants from the Canadian Institutes of Health Research (PJT-162083), Reuben and Helene Dennis Scholar and Sun Life Financial New Investigator Award for Diabetes Research from Banting &#38; Best Diabetes Centre (BBDC) and Natural Sciences and Engineering Research Council (NSERC) of Canada (RGPIN-2016-06610). R.Y.K. was supported by a fellowship from the University of Ottawa Cardiology Research Endowment Fund. J.H.L. was supported by the NSERC Doctoral Scholarship and Ontario Graduate Scholarship. Y.O. was supported by UOHI Endowed Graduate Award and Queen Elizabeth II Graduate Scholarship in Science and Technology.

All methods and protocols here have been approved by Animal Care Committees in The Animal Care and Veterinary Service (ACVS) of the University of Ottawa and The Centre for Phenogenomics (TCP) and conform to the standards of the Canadian Council on Animal Care. It should be noted that all procedures described here should be performed under institutional and governmental approval as well as by staff who are technically proficient. All mice were housed in standard vented cages in temperature- and humidity-controlled rooms with 12 h/12 h light/dark cycles (21&#8211;22 &#176;C, 30%&#8211;60% humidity for normal housing) and free access to water. Male C57BL/6J and ob/ob mice were obtained from the Jackson Laboratory.
1. 2:1 Isocaloric IF Regimen
<ol>
	<li>For lean and diet-induced obesity mouse models, prepare either a normal diet (17% fat, ND) or high fat diet (45% fat, HFD). 
	NOTE: 60% HFD can be used to induce severe diet-induced obesity; yet, due to the softness of the food pellet, it is relatively difficult to accurately measure daily food intake. An automated continuous measurement system can improve versatility for multiple types of diets.</li>
	<li>Measure baseline body weight and body composition of each mouse at 7 weeks of age using a scale and EchoMRI, respectively. 
	NOTE: Refer to section 3 for body composition measurement.</li>
	<li>Based on body weight and body composition results, randomly and equally divide 7 week-old male C57BL/6J mice into two groups: ad libitum (AL) and intermittent fasting (IF) groups.</li>
	<li>Place two to three mice per cage and ensure free access to drinking water. 
	NOTE: The number of mice per cage can affect food intake behavior. It is recommended to maintain an equal number of mice per cage in all groups during the study.</li>
	<li>Provide 1 week of acclimation to the new cage environment and diet before starting the IF regimen.</li>
	<li>Fasting period: move mice to a clean cage with fresh bedding at 12:00 PM. Do not add food for the IF group, while providing a weighed amount of food to the AL group. 
	NOTE: For each fasting cycle, it is important to change cages for both AL and IF groups to ensure that both groups are exposed to the same amount of handling time.</li>
	<li>After 24 h, measure the weights of mice in both groups and leftover food in AL cages. 
	NOTE: Make sure to include the weight of food crumbs on the food hopper and bottom of the cage, especially when using HFD, as mice often remove small pellets or fragments of food from the hopper and keep them near nest sites. The average energy intake per mouse at the end of each 2:1 cycle (3 days) is around 35 kcal, equivalent to ~10 g for a normal diet (3.3 kcal/g) and ~7 g for HFD (4.73 kcal/g).</li>
	<li>Feeding period: provide a weighed amount of food at 12:00 PM for both AL and IF groups.</li>
	<li>After 48 h of providing the food, measure the weight of leftover food and mice.</li>
	<li>Repeat steps 1.6&#8211;1.10 for the duration of the study (e.g., 16 weeks).</li>
</ol>
2. Pair-feeding (PF) Control Group
NOTE: For an IF experiment in which altered feeding behavior is observed in a mouse model (e.g., hyperphagia in ob/ob mice), it is necessary to have a pair-feeding group as a control for proper calorie-independent comparison to IF.
<ol>
	<li>For the PF control group, stagger the experiment schedule such that the same amount of food consumed by the IF group is offered to the PF group (Figure 2).</li>
	<li>Measure the amount of food consumed by the IF group over 2 days of refeeding period.</li>
	<li>Divide this amount of consumed food in the IF group evenly into three proportions and provide it daily to the PF group at 12:00 PM. 
	NOTE: Providing an equal amount of food daily is critical. In the case of mice with hyperphagia, if the pair-fed mice are provided with an amount of food less than their voluntary consumption at once, they will likely consume all provided food and become effectively fasted. This may then prevent proper comparison to IF-treated mice and confound the result.</li>
	<li>Repeat steps 2.1&#8211;2.3 for the duration of the study.</li>
</ol>
3. Body Composition Analysis
NOTE: Since long-term IF affects body weight in mice, body composition can be measured at appropriate cycles (e.g., every 3 or 4 cycles) using a body composition analyzer to quantify fat and lean mass in live, non-anesthetized mice.
<ol>
	<li>Turn on the body composition analyzer. 
	NOTE: Before starting the program, leave the machine on for at least 2&#8211;3 h to warm up.</li>
	<li>Run a system test on the body composition analyzer to test its measurement accuracy. If necessary, calibrate the system using canola oil and water samples.</li>
	<li>Measure the body weight of each mouse.</li>
	<li>Place the mouse in a small animal cylindric holder.</li>
	<li>Insert a delimiter to constrain physical movement of the mouse during the measurement and place the holder into the body composition analyzer.</li>
	<li>Run the scanning program. 
	NOTE: It takes approximately 90&#8211;120 s to analyze.</li>
	<li>After measurement, remove the holder from the equipment and bring the mouse back to the cage. 
	NOTE: A more detailed protocol can be found in a previous publication9.</li>
</ol>
4. Glucose and Insulin Tolerance Tests
<ol>
	<li>For glucose tolerance test (GTT), measure body weight and body composition of each mouse before subjecting to fasting and mark the tail with a permanent marker for easy and rapid indexing.</li>
	<li>Place mice in new cages without food at 7:00 PM for overnight fasting. 
	NOTE: Overnight fasting is the standard protocol, yet due to mouse physiology (e.g., increased glucose utilization after prolonged fasting10,11), shorter fasting (~6 h) can be used as described for ITT.</li>
	<li>After fasting 14&#8211;16 h (9:00 AM in the following morning), measure body weight and body composition of each mouse and calculate the amount of glucose dosage based on body weight. 
	NOTE: To avoid overestimation of glucose intolerance in obese mice, lean mass obtained from the body composition analysis can be used to calculate glucose dosage12,13.</li>
	<li>For each mouse, cut the tip of the tail (0.5&#8211;1.0 mm) using clean surgical scissors. After wiping off the first drop of blood, draw a fresh drop of blood from the tail and measure baseline fasting blood glucose level with the glucometer. 
	NOTE: Additional tail cutting is not required for every blood glucose measurement during GTT or ITT. The wound can be freshened by abrading it with gauze to draw a drop of blood.</li>
	<li>Subject mice to an intraperitoneal (i.p.) injection of glucose (1 mg/g of body weight). 
	NOTE: Based on the objective of an experiment (e.g., examining incretin effects), oral administration of glucose can be performed by oral gavage. The protocol for oral GTT (OGTT) can be found in another study14.</li>
	<li>Measure blood glucose from the tail at 0, 5, 15, 30, 60, and 120 min post-glucose injection.</li>
	<li>After finishing the GTT, provide a sufficient amount of food.</li>
	<li>For the insulin tolerance test (ITT), remove food at 9:00 AM. 
	NOTE: Since both GTT and ITT are stress-inducing experiences for mice that can elevate blood glucose levels and change physiology, it is recommended to perform ITT after providing at least 2&#8211;3 days of recovery after GTT experiment.</li>
	<li>After fasting for 6 h (3:00 PM), measure baseline blood glucose from the tail as described in step 4.4.</li>
	<li>Subject mice to i.p. injection of insulin (0.65 mU/g of body weight).</li>
	<li>Measure blood glucose from the tail at 0, 15, 30, 60, 90, and 120 min post-insulin injection.</li>
	<li>After finishing ITT, provide a sufficient amount of food.</li>
</ol>
5. Indirect Calorimetry
NOTE: Energy metabolism of IF-treated mice can be further evaluated through indirect calorimetry over a single cycle of IF. This will measure oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory exchange ratio (RER), and heat (kcal/h).
<ol>
	<li>Turn on the power of the indirect calorimeter system at least 2 h before running the experiment. 
	NOTE: This system warm-up is important for accurate measurement.</li>
	<li>Prepare cages with clean bedding, fill water bottles, and add the pre-weighed amount of chow to the food hoppers.</li>
	<li>Check the condition of the Drierite and lime soda. If a color indicator of the Drierite appears pink, which indicates that the Drierite has absorbed a high amount of moisture, it is necessary to replace or top with fresh Drierite.</li>
	<li>Calibrate the system using a gas with the specific composition (0.5% CO2, 20.5% O2).</li>
	<li>Measure body weight and body composition of each mouse, which will be used to normalize VO2 and VCO2 data.</li>
	<li>Gently place one mouse per cage.</li>
	<li>Assemble metabolic cages, place them in the temperature-controlled environment chamber, and connect to gas lines and activity sensor cable.</li>
	<li>After setting up the experiment profile by adding appropriate experimental parameters using the software, run the program for measurement. The purpose of the first day&#39;s measurement is to provide a period of acclimatization and measure baseline energy metabolism.</li>
	<li>At 12:00 PM the following day, subject mice to 24 h of fasting by removing food and crumbs from the hopper and bottom of the cage. If necessary, replace with clean bedding.</li>
	<li>After 24 h, add the pre-weighed amount of chow to the food hopper for the refeeding period.</li>
	<li>Continue to measure for the next 48 h. Check regularly whether the system is running without hardware or software interruption.</li>
	<li>After completing measurement, terminate the program and bring mice back to their original cages. Measure the amount of leftover food to examine food intake.</li>
	<li>The detailed protocol for indirect calorimetry can be found in a previous study9.</li>
</ol>

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The authors have nothing to disclose.

It has been well-documented that IF provides beneficial health effects on various diseases in both humans and animals8,15,16,17,18,19. Its underlying mechanisms, such as autophagy and gut microbiome, have recently been elucidated. The presented protocol describes an isocaloric 2:1 IF regimen in mice for investigating calorie-independent metabolic benefits of IF against diet-induced obesity and associated metabolic dysfunction. Unlike the alternate day fasting (1:1 IF) protocol that results in a reduction in overall caloric intake6,7, providing 1 more day of refeeding in the 2:1 IF regimen enables maintenance of an isocaloric condition in wild-type mice.
Additionally, compared to 1:1 IF, the 2:1 IF regimen may reduce possible fasting-mediated stress or torpor in mice20 and is also comparable to a popular dietary method, the 5:2 diet2. Although its effects have not been tested, the regimen can be modified by providing additional days of refeeding (e.g., 3:1 or 4:1 IF). Moreover, this protocol presented can be easily adjusted to an hourly-scale called time-restricted feeding (TRF), in which access to food is limited to 8 h per day during the active phase21, which is known to achieve an isocaloric diet regimen and provide metabolic benefits against HFD-induced obesity and diabetes19,21,22.
As shown in the feeding analysis (Figure 1B), hyperphagic behavior immediately after 24 h of fasting decreases gradually in wild-type mice, which enables isocaloric IF. However, this isocaloric condition cannot be attained in ob/ob mice, as they lack leptin signaling-mediated satiety and energy metabolism, leading to a continuous hyperphagic phenotype23,24. Therefore, before performing an IF experiment, it is recommended to examine feeding behavior of the mouse model of interest. To examine the effects of IF using a hyperphagic mouse model (e.g., ob/ob, db/db, Sim1+/-, MC4R-/-)24,25,26, as described in this protocol, employment of a pair-feeding group as an isocaloric experimental control is important for making proper comparisons. It also requires careful planning when testing a mouse model with a hypophagic phenotype (e.g., melanin-containing hormone KO mice)27.
An important factor to consider for IF studies is housing temperature, which affects various physiological and behavioral parameters in mice. Particularly, cold exposure (4&#8211;6 &#176;C) significantly increases energy intake to maintain core body temperature28. In contrast, in thermoneutral conditions (30 &#176;C) under which heat gain is balanced by heat loss, reductions in food consumption is markedly reduced8. With respect to metabolic outcomes, cold exposure induces adipose thermogenesis, which is hampered by thermoneutral condition. Therefore, it is expected that housing temperature influences the metabolic phenotypes of IF and appropriate feeding:fasting ratio to achieve isocaloric IF.
Indeed, it has been previously demonstrated that isocaloric 2:1 IF can be achieved in thermoneutral conditions, leading to improved metabolic health in diet-induced obesity and metabolic dysfunction without differences in food intake between IF and AL groups8. However, isocaloric IF may not be achievable with 2:1 ratio at cold temperatures because mice under cold exposure will show a hyperphagic phenotype, which leads to underfeeding in the IF group. Since cold exposure and IF display comparable metabolic outcomes and mechanisms (i.e., adipose thermogenesis and improved glucose homeostasis) that help fight obesity, there is interest in combining these two interventions to maximize metabolic impact. Therefore, to properly test this, performing the feeding analysis before running an IF experiment and utilizing a pair-feeding control group under cold exposure are recommended.
Other factors that may potentially affect the outcomes of IF studies include housing density. Similar to the previous study, which showed reduced food consumption in more densely housed mice29, mice from a cage of five consumed significantly less food than those from a cage of two (unpublished results). In addition, it has been demonstrated that housing density significantly affects ambient temperature, as the temperature inside a cage housing five mice is 1&#8211;2 &#176;C higher than those housing one to two mice30. Although this study concluded that housing density did not significantly affect food intake (examined for 5 weeks), in an IF study lasting 12&#8211;16 weeks, temperature inside the cage affected by housing density may still influence food intake and energy metabolism. Together, it is important to keep the same number of mice housed in a cage and minimize changing the number per cage over the course of a study.
In summary, this report shows a simple and reproducible protocol for testing isocaloric 2:1 IF in mice. Although the current study is focused on metabolic benefits of IF in diet-induced obesity and metabolic dysfunction, it can be easily adapted to investigate the protective and therapeutic effects of isocaloric IF against other conditions, such as cardiovascular and neurological diseases.

Modern lifestyle is associated with longer daily food intake time and shorter fasting periods1. This contributes to the current global obesity epidemic, with metabolic disadvantages seen in humans. Fasting has been practiced throughout human history, and its diverse health benefits include prolonged lifespan, reduced oxidative damage, and optimized energy homeostasis2,3. Among several ways to practice fasting, periodic energy deprivation, termed intermittent fasting (IF), is a popular dietary method that is widely practiced by the general population due to its easy and simple regimen. Recent studies in preclinical and clinical models have demonstrated that IF can provide health benefits comparable to prolonged fasting and caloric restriction, suggesting that IF can be a potential therapeutic strategy for obesity and metabolic diseases2,3,4,5.
IF regimens vary in terms of fasting duration and frequency. Alternate day fasting (i.e., 1 day feeding/1 day fasting; 1:1 IF) has been the most commonly used IF regimen in rodents to study its beneficial health impacts on obesity, cardiovascular diseases, neurodegenerative diseases, etc.2,3. However, as shown in previous studies6,7, and further mechanistically confirmed in our energy intake analysis8, 1:1 IF results in underfeeding (~80%) due to the lack of sufficient feeding time to compensate for energy loss. This makes it unclear whether the health benefits conferred by 1:1 IF are mediated by calorie restriction or modification of eating patterns. Therefore, a new IF regimen has been developed and is shown here, comprising of a 2 day feeding/1 day fasting (2:1 IF) pattern, which provides mice with sufficient time to compensate for food intake (~99%) and body weight. These mice are then compared to an ad libitum (AL) group. This regimen enables examination of the effects of isocaloric IF in the absence of caloric reduction in wild-type mice.
In contrast, in a mouse model that exhibits altered feeding behavior, AL feeding may not be a proper control condition to compare and examine the effects of 2:1 IF. For example, since ob/ob mice (a commonly used genetic model for obesity) exhibit hyperphagia due to the lack of leptin regulating appetite and satiety, those with 2:1 IF exhibit ~20% reduced caloric intake compared to ob/ob mice with AL feeding. Thus, to properly examine and compare the effects of IF in ob/ob mice, a pair-feeding group as a suitable control needs to be employed.
Overall, a comprehensive protocol is provided to perform isocaloric 2:1 IF, including use of a pair-feeding control. It is further demonstrated that isocaloric 2:1 IF protects mice from high fat diet-induced obesity and/or metabolic dysfunction in both wild-type and ob/ob mice. This protocol can be used to examine the beneficial health impacts of 2:1 IF on various pathological conditions including neurological disorders, cardiovascular diseases, and cancer.

<table><tbody><tr><td>Comprehensive Lab Animal Monitoring System (CLAMS)</td><td>Columbus Instruments</td><td></td><td>Indirect calorimeter</td></tr><tr><td>D-(+)-Glucose solution</td><td>Sigma-Aldrich</td><td>G8769</td><td>For GTT</td></tr><tr><td>EchoMRI 3-in-1</td><td>EchoMRI</td><td>EchoMRI 3-in-1</td><td>Body composition analysis</td></tr><tr><td>Glucometer and strips</td><td>Bayer</td><td>Contour NEXT</td><td>These are for GTT and ITT experiments</td></tr><tr><td>High Fat Diet (45% Kcal% fat)</td><td>Research Diets Inc.</td><td>#D12451</td><td>3.3 Kcal/g</td></tr><tr><td>High Fat Diet (60% Kcal% fat)</td><td>Research Diets Inc.</td><td>#D12452</td><td>4.73 Kcal/g</td></tr><tr><td>Insulin</td><td>El Lilly</td><td>Humulin R</td><td>For ITT</td></tr><tr><td>Mouse Strain: B6.Cg-Lepob/J</td><td>The Jackson Laboratory</td><td>#000632</td><td>Ob/Ob mouse</td></tr><tr><td>Mouse Strain: C57BL/6J</td><td>The Jackson Laboratory</td><td>#000664</td><td></td></tr><tr><td>Normal chow (17% Kcal% fat)</td><td>Harlan</td><td>#2918</td><td></td></tr><tr><td>Scale</td><td>Mettler Toledo</td><td></td><td>Body weight and food intake measurement</td></tr></tbody></table>

assessment of the metabolic effects of isocaloric 2:1 intermittent fasting in mice

Intermittent fasting (IF), a dietary intervention involving periodic energy restriction, has been considered to provide numerous benefits and counteract metabolic abnormalities. So far, different types of IF models with varying durations of fasting and feeding periods have been documented. However, interpreting the outcomes is challenging, as many of these models involve multifactorial contributions from both time- and calorie-restriction strategies. For example, the alternate day fasting model, often used as a rodent IF regimen, can result in underfeeding, suggesting that health benefits from this intervention are likely mediated via both caloric restriction and fasting-refeeding cycles. Recently, it has been successfully demonstrated that 2:1 IF, comprising 1 day of fasting followed by 2 days of feeding, can provide protection against diet-induced obesity and metabolic improvements without a reduction in overall caloric intake. Presented here is a protocol of this isocaloric 2:1 IF intervention in mice. Also described is a pair-feeding (PF) protocol required to examine a mouse model with altered eating behaviors, such as hyperphagia. Using the 2:1 IF regimen, it is demonstrated that isocaloric IF leads to reduced body weight gain, improved glucose homeostasis, and elevated energy expenditure. Thus, this regimen may be useful to investigate the health impacts of IF on various disease conditions.

Figure 1 shows the feeding analyses after 24 h fasting and the comparison between 1:1 and 2:1 intermittent fasting. A 24 h fasting period resulted in a ~10% reduction in body weight, which was fully recovered after 2 days of refeeding (Figure 1A). A 24 h fasting period induced hyperphagia during the subsequent 2 days of refeeding (Figure 1B). Nevertheless, the comparison of energy intake between 1:1 alternative day fasting and 2:1 intermittent fasting revealed that the 1 day of the refeeding period in 1:1 IF was not sufficient (~80%) to compensate for the caloric loss by fasting, compared to the AL condition (Figure 1C). On the other hand, 99% of energy intake was fully compensated during 2 days of refeeding in 2:1 IF. This regimen enables examination of the effects of isocaloric IF that are independent of caloric intake difference.
Figure 2 illustrates a schematic timeline for the isocaloric 2:1 IF and PF regimens. To minimize the differences in caloric intake, an observation made in alternate day fasting6,7, this protocol established a new IF regimen comprising of 2 day feeding and 1 day fasting periods (2:1 IF)8, which enabled the examination of the health effects of isocaloric IF in wild-type mice. However, in ob/ob mice, which exhibited hyperphagic behavior, 2:1 IF-treated ob/ob mice showed a 21% caloric intake reduction, compared to ob/ob AL mice15. Since this prevents a proper caloric-independent comparison, a PF control group that maintained the same caloric intake as IF-treated ob/ob mice was used. Briefly, the total amount of food consumed during 2 days of feeding in 2:1 IF mice were divided equally into three daily amounts, then provided to the PF group.
For a comprehensive overview on the metabolic outcomes of 2:1 IF, we compared the effects of AL, IF, and PF in body weight, food intake and body composition in wild-type and ob/ob mice under normal diet (ND) and HFD. Compared to AL, IF treatment led to lower body weight increase in ND-fed and HFD-fed WT mice without significant differences in food intake (Figure 3A,B). Body composition analysis revealed that IF specifically reduced fat mass without changes in lean mass in wild-type mice (Figure 3C). It is possible that a slightly, albeit not significantly, lower accumulated energy intake over 16 weeks of the IF program could result in reduced body weight gain of IF animals. However, IF experiment with the pair-feeding regimen confirmed that the decreased body weight gain by IF was not due to altered energy intake (Figure 3D,E). Unlike wild-type animals, body weight of ob/ob mice subjected to IF (Ob-IF) was lower than that of Ob-AL mice (Figure 3G). This is due to hyperphagia (excessive eating) of ob/ob mice, leading to mildly higher (21%) food intake in AL mice, compared to IF-treated animals (Figure 3H). Therefore, to specifically examine the metabolic effect of IF in a caloric-independent manner, a pair-feeding control group was employed. However, unlike wild-type mice8, Ob-PF mice were indistinguishable compared to Ob-IF mice in body weights and body composition15 (Figure 3I). These results suggest that leptin is likely implicated in isocaloric IF-mediated body weight reduction in mice.
The major metabolic benefit conferred by isocaloric IF is the improved glucose homeostasis. As shown in Figure 4A,B,C,D, HFD-IF mice exhibited a significant improvement in glucose homeostasis. GTT showed that blood glucose is more rapidly cleared in IF-treated mice, while ITT revealed higher insulin sensitivity in HFD-IF mice, compared to HFD-AL or HFD-PF mice. Unexpectedly, despite the failures in IF-mediated weight reduction, Ob-IF animals exhibited significantly improved glucose handling with smaller glucose excursions in GTT, compared to Ob-PF mice (Figure 4E), whereas insulin sensitivity was indistinguishable between Ob-IF and Ob-PF mice (Figure 4F). This improved glucose homeostasis in Ob-IF mice is likely mediated by increases in plasma level of glucagon-like peptide-1 (GLP-1) and glucose-stimulated insulin secretion (data not shown)15. Overall, by using this 2:1 IF protocol and proper caloric-independent PF control, we showed the metabolic benefits of isocaloric IF in wild-type and ob/ob mice.
One of the metabolic effects of IF in wild-type mice is higher total O2 consumption, used to estimate the energy expenditure (Figure 5A,B). This elevation in O2 consumption was found only during feeding period in IF mice, but not fasting period, compared to AL mice. The increased energy expenditure was largely mediated by adipose thermogenesis, such as browning of white adipose tissues and activation of brown adipose tissue (data not shown)8,16. IF-mediated adipose thermogenesis would presumably explain how wild-type mice subjected to IF exhibited the reduced body weight gain with no difference in food intake, compared to AL mice. On the other hand, IF failed to increase O2 consumption in ob/ob mice (Figure 5C-D), and even led to a reduction in energy expenditure during fasting period. Consistently, IF-induced adipose thermogenesis was completely abolished in ob/ob mice (data not shown). These data suggest a possible limitation of IF as it may work differently for individuals with different genetic and environmental backgrounds.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60174/60174fig1a.jpg" /> 
Figure 1: Feeding analyses after 24 h fasting and comparison between 1:1 and 2:1 IF. (A) Daily body weight changes of mice before and after 24 h fasting (n = 10). (B) Daily energy intake before and after 24 h fasting (n = 5 cages; 2 mice per cage). (C) Comparison of energy intake between alternate day fasting (i.e., 1 day feeding/1 day fasting, 1:1 IF) and 2:1 intermittent fasting (i.e., 2 day feeding/1 day fasting). In the 1:1 IF regimen, only ~80% of food intake was compensated during the subsequent 1 day of refeeding compared to food intake over 2 days of feeding. On the other hand, 99% of energy intake was achieved when 2 days of refeeding was given, compared to that over 3 days of feeding. Data are expressed as mean &#177; SEM. This figure was reproduced with permission from Kim et al.8. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60174/60174fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60174/60174fig2a.jpg" /> 
Figure 2: Schematic illustration of the isocaloric 2:1 IF regimen. For PF control, the amount of food consumed during the 2 days of feeding by IF-treated mice is divided into three equal portions, which is then provided daily to PF mice during the next cycle. AL = ad libitum; PF = pair-feeding. Part of this figure was reproduced with permission from Kim et al.8. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60174/60174fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60174/60174fig3a.jpg" /> 
Figure 3: Comparison of AL, IF, and PF effects on body weight, food intake, and body composition between wild-type and ob/ob mice. (A,B,C) Body weight, food intake, and body composition in AL or IF-treated wild-type mice under normal diet (ND) or high fat diet (HFD) during 16 weeks of IF regimen. Data are expressed as mean &#177; SEM. (ND-AL: n = 7; ND-IF: n = 8; HFD-AL: n = 7; and HFD-IF: n = 8); one- or two-way ANOVA with Student-Newman-Keuls post-hoc analysis; **p &#60; 0.01 vs. HFD-AL. (D,E,F) Body weight, food intake, and body composition in PF vs. IF mice fed with high fat diet (HFD) during 12 weeks of IF regimen. (PF: n = 6 and IF: n = 6); two-tailed unpaired Student&#39;s t-test; *p &#60; 0.05 vs. HFD-PF; NS = not significant. (G,H,I) Body weight, food intake, and body composition in AL, PF, or IF-treated ob/ob mice fed with normal chow (Ob-AL: n = 4; Ob-PF: n = 7; Ob-IF: n = 6); Ob-AL vs. Ob-PF: *p &#60; 0.05; Ob-AL vs. Ob-IF: *p &#60; 0.05; Ob-PF vs. Ob-IF. Panels A&#8211;F were reproduced with permission from Kim et al.8. Panels G&#8211;I were reproduced with permission from Kim et al.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60174/60174fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60174/60174fig4a.jpg" /> 
Figure 4: Improved glucose homeostasis by IF in both wild-type and ob/ob mice. (A,B) Intraperitoneal GTT and ITT in HFD-AL and HFD-IF wild-type mice after 16 weeks of IF regimen. The inset shows area under curve (AUC); *p &#60; 0.05 vs. HFD-AL. (C,D) GTT and ITT in HFD-PF compared to HFD-IF wild-type mice after 12 weeks of IF regimen. The inset shows AUC; *p &#60; 0.05 vs. HFD-PF. (E,F) GTT and ITT in Ob-IF compared to Ob-PF mice after 16 weeks of IF regimen. The inset shows AUC (*p &#60; 0.05 vs. Ob-PF). Panels A&#8211;D were reproduced with permission from Kim et al.8. Panels E and F were reproduced with permission from Kim et al.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60174/60174fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60174/60174fig5a.jpg" /> 
Figure 5: Energy expenditure analysis in IF-treated wild-type and ob/ob mice. (A) Traces of O2 consumption during one cycle of 2:1 IF in wild-type mice (i.e., 1 day fasting followed by 2 days of feeding). (B) Average of O2 consumption per hour during fasting, feeding, and one cycle of 2:1 IF. Data are expressed as mean &#177; SEM (HFD-AL: n = 6; and HFD-IF: n = 12); *p &#60; 0.05 vs. HFD-AL. (C) O2 consumption traces of ob/ob mice during one cycle of 2:1 IF. (D) Average of O2 consumption per hour during fasting, feeding, and one cycle of 2:1 IF (Ob-PF: n = 7; Ob-IF: n = 6); *p &#60; 0.05 vs. Ob-PF. Panel B was reproduced with permission from Kim et al.8. Panels C and D were reproduced with permission from Kim et al.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60174/60174fig5alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice at JoVE.com

Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice

The current article describes a detailed protocol for isocaloric 2:1 intermittent fasting to protect and treat against obesity and impaired glucose metabolism in wild-type and ob/ob mice.

Intermittent fasting (IF), a dietary intervention involving periodic energy restriction, has been considered to provide numerous benefits and counteract ...

assessment-metabolic-effects-isocaloric-21-intermittent-fasting

Nanyang Technological University

Research

JoVE Journal

Biology

8.8K Views.  University of Ottawa Heart Institute. Twee tot een intermitterend vasten regime bestaat uit een dag van vasten, gevolgd door twee dagen van voeding maakt het mogelijk om de effecten van dieetinterventies waarbij periodieke energiebeperking in plaats van de effecten van calorie-inname te bestuderen. In tegenstelling tot alternatieve dag vasten, wat resulteert in onder het voeden van twee tot een intermitterende vasten biedt muizen met voldoende tijd om volledig te compenseren voor het energieverlies en het verminderen van lichaams...

Video: Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can produce a rewarding effect that strengthens action-outcome associations and reinforces future behavior directed at obtaining these foods. Increasing evidence that the rewarding effects of energy-dense foods play a profound role in overeating and the development of obesity has heightened interest in studying the genes, molecules and neural circuitry that modulate food reward1,2. The rewarding impact of different stimuli can be studied by measuring the willingness to work to obtain them, such as in operant conditioning tasks3. Operant models of food reward measure acquired and voluntary behavioral responses that are directed at obtaining food. A commonly used measure of reward strength is an operant procedure known as the progressive ratio (PR) schedule of reinforcement.4,5 In the PR task, the subject is required to make an increasing number of operant responses for each successive reward. The pioneering study of Hodos (1961) demonstrated that the number of responses made to obtain the last reward, termed the breakpoint, serves as an index of reward strength4. While operant procedures that measure changes in response rate alone cannot separate changes in reward strength from alterations in performance capacity, the breakpoint derived from the PR schedule is a well-validated measure of the rewarding effects of food. The PR task has been used extensively to assess the rewarding impact of drugs of abuse and food in rats (e.g.,6-8), but to a lesser extent in mice9. The increased use of genetically engineered mice and diet-induced obese mouse models has heightened demands for behavioral measures of food reward in mice. In the present article we detail the materials and procedures used to train mice to respond (lever-press) for a high-fat and high-sugar food pellets on a PR schedule of reinforcement. We show that breakpoint response thresholds increase following acute food deprivation and decrease with peripheral administration of the anorectic hormone leptin and thereby validate the use of this food-operant paradigm in mice.

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can ...

Neuroscience

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill. Our methodology also includes protocols for treadmill assessment. Mice are initially acclimatized to the treadmill and then given baseline flat and uphill treadmill assessments. Exercise sessions begin with a 3 min warm-up, then three intervals of 1 min at a fast pace, followed by 1 min at an active recovery pace. Following these intervals, the mice are given a final segment that starts at the fast pace and accelerates for 1 min. The HIIT protocol is individually tailored as the speed and intensity for each mouse are determined based upon initial anaerobic assessment scores. Additionally, we detail the conditions for increasing or decreasing the intensity for individual mice depending on performance. Finally, intensity is increased for all mice every two weeks. We previously reported in this protocol enhanced physical performance in aged male mice and here show it also increases treadmill performance in aged female mice. Advantages of our protocol include low administration time (about 15 min per 6 mice, 3 days a week), strategy for individualizing for mice to better model prescribed exercise, and a modular design that allows for the addition or removal of the number and length of intervals to titrate exercise benefits.

Short session (&#8804;10 min) high intensity interval training (HIIT) is emerging as an alternative to longer exercise modalities, yet the shorter variants are rarely modeled in animal studies. Here, we describe a 10 min, 3 day a week, uphill treadmill HIIT protocol that enhances physical performance in male and female aged mice.

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, ...

Behavior

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

Biochemistry

Investigating Binge Eating Disorder with a Palatable Food Access Mouse Model

Intermittent Binge-Intake Model in Mice

Obesity affects one in eight individuals globally. Overeating, especially high-calorie foods, plays a significant role in obesity. Binge eating disorder (BED) is a complex condition caused by a combination of biological, psychological, and social factors. It involves excessive consumption of high-calorie foods in a short period and is often linked to anxiety and cravings.
Here, we present a protocol employing continuous and intermittent access to a new highly palatable food (HPF) -- M&amp;M&#39;s -- to develop a mouse model for investigating binge eating disorder (BED). The HPF, chosen for its high fat and sugar content, represents an optimal food source to study due to its strong palatability, which makes it particularly suited for examining compulsive eating behaviors. Using C57BL/6 mice, we provided continuous or intermittent access to M&amp;M&#39;s while allowing unrestricted access to standard chow and water. By the 8th day, the mice in the intermittent access group exhibited pronounced binge-eating behaviors, which persisted through the 26th day. These mice also consumed significantly more calories -- predominantly from the confectionary -- than those in the continuous access and control groups. The contrast between continuous and intermittent access underscores the critical role of feeding schedules in promoting the overconsumption of palatable foods.
Furthermore, behavioral assessments revealed intermittent access to the HPF-induced anxiety-like behaviors, highlighting the psychological impact of access patterns on both eating behavior and emotional states. By incorporating these two distinct feeding paradigms, this study offers valuable insights into how the availability of highly palatable foods can exacerbate binge-eating tendencies. This model provides a more realistic approach to studying binge-eating behaviors and their metabolic consequences. It highlights the importance of standardized models in biomedical research. Our findings offer insights into the physiological and neural mechanisms underlying BED, which could pave the way for developing effective therapeutic interventions for BED and obesity.

Author Spotlight: Accessible M&amp;M-Based Mouse Model for Investigating Binge Eating Disorder - Insights into Eating Behaviors, Anxiety, and Neural Mechanisms

A binge-eating model was established in mice using intermittent access to M&amp;M's (a highly palatable food made of fat and sugar). Mice with intermittent access to this high-fat, high-sugar food showed stress-like behaviors, increased caloric intake, and a preference for that food over standard chow compared to mice with continuous or no access to the food.

Obesity affects one in eight individuals globally. Overeating, especially high-calorie foods, plays a significant role in obesity. Binge eating disorder ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Immunology and Infection

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Medicine

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Low-Input Metabolic Studies in Mice Cell Types

Targeted Metabolomics on Rare Primary Cells

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule intermediates. However, obtaining accurate and reliable measurements of cellular metabolism, particularly in rare cell types like hematopoietic stem cells, has traditionally required pooling cells from multiple animals. A protocol now enables researchers to measure metabolites in rare cell types using only one mouse per sample while generating multiple replicates for more abundant cell types. This reduces the number of animals that are required for a given project. The protocol presented here involves several key differences over traditional metabolomics protocols, such as using 5 g/L NaCl as a sheath fluid, sorting directly into acetonitrile, and utilizing targeted quantification with rigorous use of internal standards, allowing for more accurate and comprehensive measurements of cellular metabolism. Despite the time required for the isolation of single cells, fluorescent staining, and sorting, the protocol can preserve differences among cell types and drug treatments to a large extent.

Author Spotlight: Advancing Metabolomics Analysis of Rare Hematopoietic Stem Cells 

Here, we present a protocol to accurately and reliably measure metabolites in rare cell types. Technical improvements, including a modified sheath fluid for cell sorting and the generation of relevant blank samples, enable a comprehensive quantification of metabolites with an input of only 5000 cells per sample.

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule ...

Assessing Whole-Body Lipid-Handling Capacity in Mice

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a class of many different molecules with many pathways involved in their synthesis and metabolism. A starting point for evaluating lipid hemostasis for nutrition and obesity research is needed. This paper describes three easy and accessible methods that require little expertise or practice to master, and that can be adapted by most labs to screen for lipid-metabolism abnormalities in mice. These methods are (1) measuring several fasting serum lipid molecules using commercial kits (2) assaying for dietary lipid-handling capability through an oral intralipid tolerance test, and (3) evaluating the response to a pharmaceutical compound, CL 316,243, in mice. Together, these methods will provide a high-level overview of lipid handling capability in mice.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...

Determining Basal Energy Expenditure and the Capacity of Thermogenic Adipocytes to Expend Energy in Obese Mice

Energy expenditure measurements are necessary to understand how changes in metabolism can lead to obesity. Basal energy expenditure can be determined in mice by measuring whole-body oxygen consumption, CO2 production, and physical activity using metabolic cages. Thermogenic brown/beige adipocytes (BA) contribute significantly to rodent energy expenditure, particularly at low ambient temperatures. Here, measurements of basal energy expenditure and total BA capacity to expend energy in obese mice are described in two detailed protocols: the first explaining how to set up the assay to measure basal energy expenditure using analysis of covariance (ANCOVA), a necessary analysis given that energy expenditure co-varies with body mass. The second protocol describes how to measure BA energy expenditure capacity in vivo in mice. This procedure involves anesthesia, needed to limit expenditure caused by physical activity, followed by the injection of beta3-adrenergic agonist, CL-316,243, which activates energy expenditure in BA. These two protocols and their limitations are described in sufficient detail to allow a successful first experiment.

This manuscript describes a protocol to measure the basal metabolic rate and the oxidative capacity of thermogenic adipocytes in obese mice.

Energy expenditure measurements are necessary to understand how changes in metabolism can lead to obesity. Basal energy expenditure can be determined in ...

Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice

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