Palatable Western-style Cafeteria Diet as a Reliable Method for Modeling Diet-induced Obesity in Rodents

Summary

This protocol describes the use of a highly palatable, western-style cafeteria diet to model overeating and obesity in rodents. Here, we provide a detailed outline of food selection, preparation and measurement, and explain methodological factors that assist in generating a robust and reproducible phenotype.

Abstract

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases. The health burden of obesity and its comorbid conditions highlight the need for better understanding of its pathogenesis, yet ethical constraints limit studies in humans. To this end externally valid models of obesity in laboratory animals are essential for the understanding of being overweight and obesity. While many species have been used to model the range of changes that accompany obesity in humans, rodents are most commonly used. Our laboratory has developed a western-style cafeteria diet that consistently leads to considerable weight gain and markers of metabolic disease in rodents. The diet exposes rodents to a variety of highly palatable foods to induce hyperphagia, modeling the modern western food environment. This diet rapidly induces weight gain and body fat accumulation in rats allowing for the study of effects of overeating and obesity. While the cafeteria diet may not provide the same control over macronutrient and micronutrient profile as purified high-fat or high-fat, high-sugar diets, the cafeteria diet typically induces a more severe metabolic phenotype than that observed with purified diets and is more in line with metabolic disturbances observed in the overweight and obese human population.

Introduction

Obesity and its related comorbidities make an enormous contribution to global health burden¹ and account for 7% of disease burden in Australia². A leading risk factor for obesity is consumption of unhealthy diets that are high in saturated fat and refined carbohydrates, and low in fiber and micronutrients³. Identifying targets for therapeutic intervention for obesity requires models that can systematically assess effects on multiple biochemical and physiological systems. Our understanding of the etiology of obesity has been advanced substantially by work using rodent models, where behavioral, metabolic and molecular effects can be studied across time under controlled conditions where environmental factors can be easily manipulated.

The cafeteria diet (CAF) model of diet-induced obesity consists of supplementing rodents' standard chow diet with a variety of palatable foods that are high in either saturated fat, refined carbohydrates, or both. Examples of these foods include cakes, sweet biscuits, and high-fat savory snacks (such as processed meats, cheese and chips). It reliably promotes hyperphagia and rapid weight gain in rodents. The key features of the model are the provision of a variety of highly palatable foods, designed to simulate the modern food environment. Access to variety increases food intake in rats over the short-term⁴ and in humans⁵ even when the foods are matched for palatability and vary only in flavor and olfactory cues^4,6. However, one study showed that providing energy- and macronutrient-matched purified diets that varied in flavor and texture had no effect on long-term body weight gain in rats⁷, suggesting that nutrient composition and distinct post-oral effects of different foods may also contribute to overeating. Exposure to multiple tastes and textures overcomes sensory-specific satiety, which describes the decrease in desire to eat a recently eaten food relative to an alternative⁵. Across many cohorts in our laboratory, we have similarly observed that the use of highly palatable foods further amplifies overeating.

This CAF diet has been used for over 40 years, since Sclafani⁸ reported that female rats exposed to an assortment of ‘supermarket foods' (marshmallows, chocolate, peanut butter, cookies, salami and cheese among them) exhibited accelerated weight gain relative to controls. This and other early studies noted that CAF-style diets appeared to accelerate weight gain more effectively than pure high-fat or high-carbohydrate diets ^8,9. Work in the 1980s characterized the macronutrient profiles¹⁰ and meal patterns¹¹ of rats fed CAF diets, and showed profound changes to fat mass and insulin levels^9,10 and thermogenesis¹². Our group has used the CAF diet to model obesity for over two decades^13,14 and during this time we have used several variants of the diet. Rats are presented with at least two sweet and two savory food items each day, in addition to regular chow and water. In recent years we have begun to supplement solid CAF foods with 10% sucrose solution. The ability to tailor the CAF diet to different experimental designs is a strength of the model.

CAF diets promote immediate hyperphagia (i.e., within the first 24 h) and steady gains in body weight and fat mass. However, a consequence of maximizing variety is that macronutrient and micronutrient intake is not controlled, a point some view as an insurmountable flaw¹⁵. Studies of diet-induced obesity more commonly use purified high-fat (HF) or combined high-fat, high-sugar (HFHS) diets, which offer precise control over nutritional content and are less labor-intensive than the CAF model, which requires daily monitoring and careful planning and execution of the schedule. The translational relevance of commercially available purified HF diets is a topic of ongoing debate, as their fatty acid profile and proportions of fat and sucrose may not align with human dietary intake¹⁶. While CAF diet does not offer the same degree of control over nutrient composition as purified diets, it aims to model the palatability and variety that characterizes food options in most modern societies.

Protocol

The protocol described here has been optimized for use in rats. While we have used the CAF diet successfully in mice^17,18, soft food grinding may introduce further error reducing the reliability of food intake measures¹⁹. This protocol is approved by the Animal Care and Ethics Committee at the University of New South Wales and complies with the Australian guidelines for the use and care of animals for scientific purposes (8th Edition) provided by the Australian National Health and Medical Research Council.

NOTE: Very few adverse effects have been observed in our short-term studies (i.e. <10 weeks ad libitum CAF access); there is no evidence of changes to general wellbeing, activity, sociability or anxiety-like behavior in rats on CAF diet²⁰. After longer intervals (>16 weeks) very occasional cardiovascular incidents have been observed in CAF-fed rats.

1. Animal Acclimatization and Housing

1. Acclimate rats to the facility for 5–7 days after arrival with free access to control diet and water. Handle rats daily beginning 24 h after arrival; CAF diet involves daily interaction with the cage so regular handling is important.
2. Ensure environmental enrichment is provided to all cages. A standard cage contains a red polymethyl methacrylate box, nesting material and a wooden chew stick, which is important because the soft foods provided within the CAF diet may result in rats developing malocclusion without access to harder items to chew.
3. Prior to commencing CAF diet, assign cages to CAF or chow groups and ensure these are matched for starting body weight by comparing the mean and range of body weights between groups and re-allocating as necessary. As lighting exposure can affect circadian rhythms, food intake and activity, ensure CAF and chow cages are distributed evenly in the colony room.
   NOTE: Rodents form social hierarchies when group-housed (especially males). The effects of social stress are partially controlled for by ensuring rats are housed with others of a similar body weight (to reduce bullying within a cage). Additionally, cafeteria diet should be evenly distributed around the cage so that all rats have access to the diet.

2. Diet Selection and Setup

1. Scheduling
   1. Ensure regular chow and water are always available and are supplemented with a minimum of two sweet and two savory items each day (an example of a weekly schedule is shown in Table 1; food options for 3 days shown in Figure 1). Optional extras are high-fat, high-sugar chow and 10% sucrose solution.
   2. Choose foods within each category that are similar in macronutrient proportions: all sweet items are relatively higher in carbohydrate than fat and all savory items are higher in fat than carbohydrate. Aim to provide similar proportions of fat, carbohydrate, sugar and protein in each daily set of CAF foods.
   3. Regularly monitor consumption throughout the experiment so that the menu can be tailored, if necessary, to sustain hyperphagia. Table 2 provides energy density and macronutrient information for several example foods, and suggested starting amounts for 200 g male Sprague Dawley rats.
   4. Avoid feeding any single food on consecutive days or too often in a week, as this may decrease intake of that food. If this occurs, omit the food from the schedule for a few days before re-incorporating.
      NOTE: Rats may display neophobia and most new foods require repeated exposures to determine whether they will be consumed. Rats do not typically prefer foods containing yeast (breads, pizza, etc.) or those with citrus or coffee flavors.
2. Wet foods
   1. Consider presenting foods higher in moisture content in a container such as a tuna tin, to avoid soiling the bedding. Empty containers should also be provided to control cages if this is done.
3. Diet sourcing
   1. Ensure that CAF foods used are staples or obtain enough for the whole experiment so that food items remain consistent and do not need to be substituted.
4. Diet storage
   1. Store foods as indicated on the packaging or at -20 °C and thaw the night before use. Store dry goods (biscuits) in an airtight container.
5. Sucrose solution (optional)
   1. Prepare sucrose solution, typically 10% (w/v) in bulk (2–5 L) and store at 4 °C when not in use.
   2. Replace sucrose bottles weekly (at a minimum) to prevent bacterial or fungal growth. Inspect bottles daily and replace if signs of growth are observed.
      NOTE: Sucrose is provided in addition to water, which is always available.

3. Cafeteria Diet Preparation

1. Defrost
   1. Take out appropriate amounts of CAF foods to thaw ~24 h prior to use. Food can be defrosted using a microwave but should not be presented while hot.
2. Daily replenishing of CAF diet
   1. Changing CAF diet foods daily ensures that variety is maximized, and cage soiling is minimized. As rats eat most of their daily intake in the initial portion of the dark cycle, schedule CAF food replenishment close to the onset of the dark schedule so that food is fresh at this time. Replenish CAF diet on a day when food intake is not being measured.
   2. Feed the chow groups in a manner that ensures that this group has a similar daily experience, as follows.
      1. Turn the water bottles around (spouts up), place the cage on the workspace and remove the lid.
      2. Lightly disturb the bedding for approximately 20 s, to simulate the process of collecting food items from the bedding of CAF cages.
      3. Place a small handful of chow pellets from the food hopper into the bedding, to equate exposure to food on the cage bedding.
      4. Return the cage lid and return to the rack, replacing water bottles only when the cage is settled in order to minimize spillage.
   3. Feeding the cafeteria groups
      1. Prepare CAF diet items into a labeled container for each cage.
      2. Turn the water bottles around (spouts up), place the cage on the workspace and remove the lid.
      3. Remove as much of the old cafeteria diet from the bedding as possible.
      4. Place fresh cafeteria diet into the cage.
      5. Close the cage and return to the rack.
      6. Replace the water and sucrose bottles.
         NOTE: To avoid exposing chow rats to CAF diet, consider feeding chow cages before CAF cages, or vice versa. Gloves should be changed between handling rats from different diet groups.
3. Top up chow, water and sucrose bottles as necessary. Doing this after other procedures is more efficient as boxes are heavier after topping up food and water.
   NOTE: CAF boxes soil more rapidly and need to be changed more often to avoid stress induced by exposure to wet bedding as well as strong smells. Inspect bedding daily and change as appropriate.

4. Food Intake Over 24 H

NOTE: Food intake measurements are conducted over a discrete 24 h period several times per week.

1. When commencing the CAF diet, measure body weight and 24 h food intake at least twice per week to monitor the effectiveness of the diet. Begin food intake measurements close to the onset of the dark phase. Aim to present the same set of CAF foods on food intake measurement days, as equating for flavor and energy densities will permit accurate monitoring of changes over time.
   NOTE: This may not be possible for studies requiring >2 measurements of food intake per week, as excessive exposures to the same foods may reduce hyperphagia over time.
2. CAF and control diet intake measures
   1. Prepare CAF diet and place into a labeled container for each cage, weighing and recording each component. Figure 1 shows a completed set of CAF foods. Table 3 consists of an example food intake monitoring sheet.
   2. Refill water, sucrose and chow levels as required.
3. For chow (control) cages
   1. Turn water bottles around (spouts up), place the cage on the workspace and remove the lid.
   2. Weigh the rats and then transfer them to a cage with fresh bedding, together with environmental enrichment.
   3. Record weight of water bottles and chow.
   4. Replace the cage lid, return the cage to rack, and then turn the water bottles around. Small signs stating ‘FOOD INTAKE’ can be added to the cage to notify attendants and researchers not to touch bottles and chow.
4. For the CAF cages:
   1. Perform the same steps as in step 4.3. Additionally scatter the pre-weighed container of CAF foods around the cage with fresh bedding before rats are transferred.
   2. Leave rats for a 24 h period with minimal disturbance. Record any unanticipated disruptions (e.g., arrival of new rats to the colony room, or changes in ambient noise).
5. Finish food intake measurement
   1. Record weight of water bottles and chow for the chow cages, carefully searching the bedding for pieces of chow. Remove the 'food intake' sign from cages once complete.
   2. Prepare fresh CAF diet for CAF cages.
   3. For each CAF cage
      1. Record weight of water, chow and sucrose.
      2. Remove cage lid and environmental enrichment.
      3. Carefully remove CAF fragments from bedding and place into separate containers. Remove the largest pieces first and then systematically sift all cage bedding from one end to the other using gentle sweeping motions.
         NOTE: Apply the same degree of care to each cage when collecting food. Record where a cage has been particularly messy—in some instances rats will grind down cake and/or biscuit into a fine powder that is difficult to collect. This can artificially inflate food intake measures.
      4. Distribute the new CAF diet around the cage, return environmental enrichment, close the cage lid and return it to the rack, removing ‘food intake’ sign.
      5. Record weight of each CAF food to nearest 0.1 g. Subtract food remaining from the initial food weight to obtain total food consumed per cage, and then divide by the number of rats in each box (thus assuming equal intake).
      6. Multiply amount consumed (g/rat) by the energy density (kJ/g) of each food provided by the manufacturer.
         NOTE: To calculate macronutrient intake we use assume energy densities of 16.7 kJ/g for carbohydrates and protein, and 37 kJ/g for fat.

Results

As shown in Figure 2A, CAF diet feeding produces a 2.5-fold increase in energy intake relative to chow controls, based on data from three cohorts of male Sprague Dawley rats, that is consistent over 6 weeks. Other studies have confirmed that this extent of hyperphagia is sustained over 10²¹ and 16²² week experiments. The weight curve (Figure 2B) indicates CAF diet feeding leads to ...

Discussion

By exposing rats to a variety of highly palatable foods high in fat and sugar, the CAF diet protocol described here provides a reliable and robust model of the so-called ‘western diet’ eaten by many people. Hyperphagia—assessed as a significant increase in energy intake relative to controls—is observed within the first 24 h of exposure, with statistically significant body weight differences seen within weeks. Thus, CAF is an effective model of diet-induced obesity for rodents.

Materials

Name	Company	Catalog Number	Comments
2-5 L plastic bottle			For preparing 10% sucrose solution, if applicable
Chopping board			Plastic is advised
Freezer			For storing CAF foods
Gordon's maintenance rodent chow	Gordon's Specialty Stockfeeds (Australia)		Maintenance diet used in our laboratory (14 kJ/g; 65% carb, 13% fat and 22% protein, as energy)
Large plastic storage boxes			All items above can be stored in containers for easy access
Large spoon			For CAF diet preparation
Microwave			For CAF diet thawing (when required)
Non-serrated knife			For CAF diet preparation
Paper towel			Important for cleaning work surfaces and the knife during CAF prep
Plastic containers			These are for weighing CAF food items on measurement days
Plastic funnel			For preparing 10% sucrose solution, if applicable
Red light			As CAF diet should be refreshed near the onset of the dark phase each day, a red light will assist when working in the dark
Tuna tins			For presenting 'wetter' CAF food items. Plastic containers may also be suitable
Weigh container x 3			Separate containers should be used to weigh rats, chow & bottles, and CAF foods
Weighing scale			Sensitivity to 0.1g is recommended
White sugar			For 10% sucrose solution, if applicable