Intermittent Binge-Intake Model in Mice

Summary

A binge-eating model was established in mice using intermittent access to M&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.

Abstract

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&M'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&M's while allowing unrestricted access to standard chow and water. By the 8^th day, the mice in the intermittent access group exhibited pronounced binge-eating behaviors, which persisted through the 26^th 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.

Introduction

According to data from the World Health Organization¹, one in eight people worldwide is obese. Although eating is a physiological necessity, overeating without a physiological need is known to be a factor associated with overweight and obesity². It is well known that humans prefer palatable foods high in calories, such as fats and sugars, over healthier foods³.

From the perspective of complexity science, eating disorders, particularly binge eating disorder (BED), are multifaceted phenomena that emerge from the interaction of various biological, psychological, and social factors exhibiting emerging behaviors such as cyclic binging patterns, complex compensatory behaviors, and dysfunctional interpersonal relationships⁴. BED is characterized by the excessive intake of high-calorie foods within a short period, approximately 2 h, according to the Diagnostic and Statistical Manual of Mental Disorders, 5th ed.⁵ (DSM-V). Additionally, BED is associated with behavioral comorbidities such as anxiety and cravings^6,7. The complexity of these disorders requires better experimental models that can capture this interplay of factors and provide a more comprehensive understanding of binge eating behavior and its implications for obesity.

The most used models for binge-like behavior in basic research have primarily been rats. These models typically use limited and intermittent access to high-fat or high-sugar foods to induce binge-like behaviors^8,9,10. An alternative approach involves providing palatable food for a short period (2 h) every 8 days, known as a cyclical model, using a highly palatable food (HPF) paste containing a mix of fats and sugars¹¹. Another method presents the HPF as cookies unpredictably¹²_. In mouse models, binge-like behaviors are often studied using the same cyclical model¹³. Another study with mice uses limited access to sucrose to induce binge-like behaviors¹⁴.

Despite extensive research on binge-like eating behaviors in rats, models for binge eating in mice remain limited. This study aims to propose a binge-like eating model using M&M's (hereafter referred to as "HPF") as an HPF according to the nutritional criteria of HPF proposed by Fazzino and colleagues¹⁵. Additionally, we aim to evaluate the rewarding properties of the HPF based on whether they are presented continuously or intermittently. In our research, we aimed to observe similarities between specific diagnostic DSM-V criteria for binge eating in humans within a murine model: consuming food within a short period (2 h), ingesting a large amount of food than usual, eating substantial amount despite being in a state of satiety, and experiencing binge episodes at the least twice a week.

The use of laboratory mouse models is an invaluable tool in biomedical research, especially for studying human diseases, as mice share 99% of their genes with humans¹⁶. Utilizing mice also offers significant advantages in understanding transcriptomic information and modifying therapeutic targets related to cellular functions and neural circuits associated with various diseases¹⁷.

Therefore, this study proposes a continuous and intermittent access model to M&M's (an HPF made of fat and sugar) to observe binge-like eating in C57BL/6 mice without standard chow or water deprivation. This model is novel and necessary for biomedical research, as it represents a better approach to studying these complex problems. Standardizing animal models is essential to obtain reproducible and comparable results, which is fundamental to advancing translational research and developing effective therapeutic interventions for BED and obesity. The approach described here will expand the scope of binge-like eating behavior research in future studies, facilitating the testing of various drugs as potential therapeutic targets for BED. The implementation of more realistic and standardized models in mice will allow for a better understanding of the complex dynamics involved in eating disorders and their metabolic consequences, thus offering new alternatives for the treatment and prevention of BED.

Protocol

All experiments were conducted according to the Mexican Official Standard of Technical Specifications for the Production and Use of Laboratory Animals (NOM-062-ZOO-1999) and in full compliance with the guidelines set forth by the Internal Committee for the Care and Use of Laboratory Animals (CICUAL) at the National Autonomous University of Mexico (UNAM).

1. Animals

1. To follow this protocol, individually house 27 C57BLC/6 male mice, aged 1 month and weighing 16-20 g, in cages under controlled environmental conditions: a 12-12 h light-dark cycle (06:00 on, 18:00 off), maintaining the temperature at 21 ± 22 °C and humidity levels at 70 ± 10%.
   NOTE: In this study, we exclusively used male mice to eliminate the effects of female hormones during binge-type intake. However, in another study¹⁸, we applied the same model to female mice and observed no differences in binge intake.

2. Habituation

1. Provide the 30-day-old mice with ad libitum access to standard chow and water. Place them in individual cages to facilitate measuring water and food consumption.
2. Provide an enriched environment, including a paper tube, to all the mice and provide wood shavings as bedding.
3. Measure body weight, food, and water consumption every 24 h for 7 days.

3. Binge eating model

1. On day 7 of the habituation period, randomly assign the rodents (N= 27) into one of the following three groups.
2. Provide all subjects with access to the HPF (nutritional content per 100: 480 kcal, 19g of fat, 70 g of carbohydrates, 5.1 g of protein, 0.13 g of salt) for 2 h (from 16:00 to 18:00) to minimize neophobia toward the HPF.
3. Continue the specified feeding conditions for each group consistently for a period of 26 days.
   1. Chow Group (CG) n = 9: provide ad libitum access to standard chow and water.
   2. Continuous Group (CoG) n = 9; provide ad libitum access to standard chow, HPF, and water.
   3. Intermittent Group (IG) n = 9: provide ad libitum access to standard chow and water but give them HPF only on specific days (1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, and 26) for 2 h (from 16:00 to 18:00 h).
4. From the first session to session 26, measure body weight, standard chow intake, palatable food intake, and water consumption for all subjects at 16:00 h and again at 18:00 h.

4. Specifical instructions

1. Place the standard chow on the cage racks and the HPF in small plastic containers. In each session, randomly place these containers at the corners of the cages (left posterior, right posterior, right anterior, and left anterior).
2. Replenish water (280-300 mL) and standard chow (10-15 g) on Mondays, Wednesdays, and Fridays.
3. Replace the HPF (4-4.5 g) daily for the CoG. For the IG, place the HPF (4-4.5 g) on Mondays, Wednesdays, and Fridays at 16:00 h. Randomly distribute the HPF by color (brown, blue, red, orange, green, and yellow), with no preference observed for any color.
4. On day 7 of habituation, give the mice assigned to the CoG group the HPF at 16:00 h. Measure all the animals for standard chow intake, body weight, and water consumption.

5. Data analysis

1. For 24 h intake measurements, record the values at 16:00 h on the previous day and 16:00 h on the current day. For instance, to evaluate the consumption of day 2, subtract the values for standard chow, the HPF, water, and body weight from day 1 from those of day 2 for the same parameters.
2. For 2 h intake measurements, record the values at 16:00 h and 18:00 h on the same day. For example, subtract the values for standard chow, the HPF, water, and body weight at 16:00 h from those at 18:00 h for the same parameters.
3. Collect all data daily at two time points (16:00 h and 18:00 h), input them into a spreadsheet, and perform kcal calculations.
   1. To calculate the kcal, convert the grams consumed of standard chow and HPF to kcal based on the energy value of each food using a proportion (e.g., standard chow: 3.02 kcal/g; HPF: 4.80 kcal/g).
4. Analyze and plot the data using the software of choice.
   1. For statistical analysis, use the Kruskal-Wallis and Mann-Whitney U tests.

6. Open field test

NOTE: A 22-day, 14:00 h, open field test was conducted according to the protocol described by Kraeuter and colleagues¹⁹. This protocol consists of placing the animals in the center of a black acrylic sheet box. The floor of the arena was white, and the grid was black. Behavior was recorded and quantified with a video tracking system. The system recorded the distance, speed, and time the animals spent in the center or periphery of the arena.

1. Preparation of the test room and open field apparatus
   1. For the arena, use a square chamber of 60 cm (length) x 60 cm (width) x 20 cm (height) made of black nonporous plastic.
   2. Clean the device before and between uses with 70% vol/vol ethanol to remove odor cues.
   3. Place the camera above the apparatus for optimal exploration view.
   4. To minimize stress, maintain temperature and humidity like the normal housing conditions. If the mice are moved from one housing room to another for the experiment, acclimate them to the new room for at least 1 h each day before use.
2. Performing the open field test
   1. To remove a single mouse from the cage, gently grasp its tail and place it in the center of the open field maze. Activate the video tracking software using the autostart feature, which detects the investigator and begins recording as soon as the investigator is no longer in focus of the camera.
   2. Allow the mouse to move freely along the maze for 5 min. During this time, the tracking software will record the movement.
   3. At the end of the trial period, remove the animal from the arena and return it to its home cage.
   4. Before cleaning the maze, visually count the feces deposited and record them for later analysis.
   5. Remove all fecal pellets and clean any urine stains. Spray the apparatus floor and walls with 70% ethanol, then wipe with a clean paper towel. Allow the ethanol solution to dry completely.
   6. After testing all animals, return to the vivarium, clean the arena, and organize all used materials.
3. Data analysis
   NOTE: The video tracking software facilitates the extraction of a range of data, such as the animals' average speed, the distance traveled, and the time spent in the center and periphery of the apparatus.
   1. Select the distance traveled and the time spent by the animals in the center and the periphery of the apparatus.
   2. Export the data from the tracking software into a spreadsheet or other preferred format for further statistical analysis.
   3. Analyze the data with the software of choice.

Results

Using HPF, we successfully established a binge-eating model in C57BL/6 mice. By day 8, binge-eating behavior (consuming excessive food for 2 h) was firmly established and maintained through day 26 (Figure 1A). During the intermittent sessions, the IG group exhibited significantly higher total kcal consumption (from both standard chow and the HPF than the CG and CoG) groups (Figure 1A, ### p < 0.0001, *** p < 0.0001, ** p < 0.001, Kruskal-Wallis test). N...

Discussion

We used the binge-eating model proposed by Corwin in 2006 as a basis for developing this protocol. However, the present study modified the variable of providing palatable food for 2 h for the CoG⁸. Instead, we offered palatable food ad libitum to CoG, as it better simulates a reality where we have 24 h access to any food. We also used M&M's as an HPF because they combine fats and sugars, unlike other binge-eating models that use either fat or sugar separately⁸

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ANY-maze Video Tracking Software	Stoelting Co.	4.73 version
C270 HD Webcam	Logitech	C270 HD	It is not necessary to use a specific video recorder; any webcam or camcorder with satisfactory image quality and resolution will suffice.
Gloves Ambiderm (black)	Ambiderm	7.50222E+12
GraphPad Prism 9	GraphPad
Laboratory Rodent Diet 5001	LabDiet	1320	https://di.facmed.unam.mx/comisiones/Composici%C3%B3n%20del%20alimento%20Laboratory%20Rodent%20Diet%205001.pdf
M&Ms 43.8g (Brown packaging)	M&M´s		Nutritional content per 100 : 480 kcal, 19g of fat, 70 g of carbohydrates, 5.1 g of protein, 0.13 g of saltIngredients: sugar, cocoa mass, skimmed milk powder, cocoa butter, lactose, starch, milk fat, palm fat, glucose syrup, shea fat, stabiliser (gum arabic), dextrin, glazing agents (beeswax, carnauba wax), colours (E100, carmine, E132, E133, E150a, E150c, E150d, E153, E160a, E160e, E162, E163, E170), beetroot concentrate, emulsifiers (soya lecithin, E445), salt, flavourings, palm kernel oil, antioxidant (E306). (May contain: peanut, hazelnut, almond). Milk chocolate contains milk solids 14% minimum. Milk chocolate contains vegetable fats in addition to cocoa butter.
Open Field maze
Polypropylene Mice Cage	Orchid Scientific	SMP 01
Rodent drinkers	Sunnypet	SP-3656
Uline Balanza-220 g x 0.01 g	unike.mx	H-9884
UniMask4	Uniseal
Plastic bottles	unike.mx	S-14487
Sanitary Bed, Sawdust for Animals	BIOINVERT	No number	10 kg