Name: assessment of the metabolic effects of isocaloric 2:1 intermittent fasting in mice
Uploaded: 2019-11-27T13:30:00
Description: Watch this Scientific Journal Video about Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice at JoVE.com

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 w...

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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-...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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...

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 ...

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.

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Biology

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for ...