The work was supported by NHMRC project grants (#568728, #150262, #1126929) to MJM.

The protocol described here has been optimized for use in rats. While we have used the CAF diet successfully in mice17,18, 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. &#60;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 (&#62;16 weeks) very occasional cardiovascular incidents have been observed in CAF-fed rats.
1. Animal Acclimatization and Housing
<ol>
	<li>Acclimate rats to the facility for 5&#8211;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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Diet Selection and Setup
<ol>
	<li>Scheduling
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Wet foods
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Diet sourcing
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Diet storage
	<ol>
		<li>Store foods as indicated on the packaging or at -20 &#176;C and thaw the night before use. Store dry goods (biscuits) in an airtight container.</li>
	</ol>
	</li>
	<li>Sucrose solution (optional)
	<ol>
		<li>Prepare sucrose solution, typically 10% (w/v) in bulk (2&#8211;5 L) and store at 4 &#176;C when not in use.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Cafeteria Diet Preparation
<ol>
	<li>Defrost
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Daily replenishing of CAF diet
	<ol>
		<li>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.</li>
		<li>Feed the chow groups in a manner that ensures that this group has a similar daily experience, as follows.
		<ol>
			<li>Turn the water bottles around (spouts up), place the cage on the workspace and remove the lid.</li>
			<li>Lightly disturb the bedding for approximately 20 s, to simulate the process of collecting food items from the bedding of CAF cages.</li>
			<li>Place a small handful of chow pellets from the food hopper into the bedding, to equate exposure to food on the cage bedding.</li>
			<li>Return the cage lid and return to the rack, replacing water bottles only when the cage is settled in order to minimize spillage.</li>
		</ol>
		</li>
		<li>Feeding the cafeteria groups
		<ol>
			<li>Prepare CAF diet items into a labeled container for each cage.</li>
			<li>Turn the water bottles around (spouts up), place the cage on the workspace and remove the lid.</li>
			<li>Remove as much of the old cafeteria diet from the bedding as possible.</li>
			<li>Place fresh cafeteria diet into the cage.</li>
			<li>Close the cage and return to the rack.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
4. Food Intake Over 24 H
NOTE: Food intake measurements are conducted over a discrete 24 h&#160;period several times per week.
<ol>
	<li>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 &#62;2 measurements of food intake per week, as excessive exposures to the same foods may reduce hyperphagia over time.</li>
	<li>CAF and control diet intake measures
	<ol>
		<li>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.</li>
		<li>Refill water, sucrose and chow levels as required.</li>
	</ol>
	</li>
	<li>For chow (control) cages
	<ol>
		<li>Turn water bottles around (spouts up), place the cage on the workspace and remove the lid.</li>
		<li>Weigh the rats and then transfer them to a cage with fresh bedding, together with environmental enrichment.</li>
		<li>Record weight of water bottles and chow.</li>
		<li>Replace the cage lid, return the cage to rack, and then turn the water bottles around. Small signs stating &#8216;FOOD INTAKE&#8217; can be added to the cage to notify attendants and researchers not to touch bottles and chow.</li>
	</ol>
	</li>
	<li>For the CAF cages:
	<ol>
		<li>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.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Finish food intake measurement
	<ol>
		<li>Record weight of water bottles and chow for the chow cages, carefully searching the bedding for pieces of chow. Remove the &#39;food intake&#39;&#160;sign from cages once complete.</li>
		<li>Prepare fresh CAF diet for CAF cages.</li>
		<li>For each CAF cage
		<ol>
			<li>Record weight of water, chow and sucrose.</li>
			<li>Remove cage lid and environmental enrichment.</li>
			<li>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&#8212;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.</li>
			<li>Distribute the new CAF diet around the cage, return environmental enrichment, close the cage lid and return it to the rack, removing &#8216;food intake&#8217; sign.</li>
			<li>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).</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

The authors declare no competing interests or disclosures.

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 &#8216;western diet&#8217; eaten by many people. Hyperphagia&#8212;assessed as a significant increase in energy intake relative to controls&#8212;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.
<p class="jove_c...

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&#39;&#160;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 cues4,6. 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 &#8216;supermarket foods&#39;&#160;(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 levels9,10 and thermogenesis12. Our group has used the CAF diet to model obesity for over two decades13,14 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.

<table><tbody><tr><td>2-5 L plastic bottle</td><td></td><td></td><td>For preparing 10% sucrose solution, if applicable</td></tr><tr><td>Chopping board</td><td></td><td></td><td>Plastic is advised</td></tr><tr><td>Freezer</td><td></td><td></td><td>For storing CAF foods</td></tr><tr><td>Gordon&#039;s maintenance rodent chow</td><td>Gordon&#039;s Specialty Stockfeeds (Australia)</td><td></td><td>Maintenance diet used in our laboratory (14 kJ/g; 65% carb, 13% fat and 22% protein, as energy)</td></tr><tr><td>Large plastic storage boxes</td><td></td><td></td><td>All items above can be stored in containers for easy access</td></tr><tr><td>Large spoon</td><td></td><td></td><td>For CAF diet preparation</td></tr><tr><td>Microwave</td><td></td><td></td><td>For CAF diet thawing (when required)</td></tr><tr><td>Non-serrated knife</td><td></td><td></td><td>For CAF diet preparation</td></tr><tr><td>Paper towel</td><td></td><td></td><td>Important for cleaning work surfaces and the knife during CAF prep</td></tr><tr><td>Plastic containers</td><td></td><td></td><td>These are for weighing CAF food items on measurement days</td></tr><tr><td>Plastic funnel</td><td></td><td></td><td>For preparing 10% sucrose solution, if applicable</td></tr><tr><td>Red light</td><td></td><td></td><td>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</td></tr><tr><td>Tuna tins</td><td></td><td></td><td>For presenting &#039;wetter&#039; CAF food items. Plastic containers may also be suitable</td></tr><tr><td>Weigh container x 3</td><td></td><td></td><td>Separate containers should be used to weigh rats, chow &amp;amp; bottles, and CAF foods</td></tr><tr><td>Weighing scale</td><td></td><td></td><td>Sensitivity to 0.1 g is recommended</td></tr><tr><td>White sugar</td><td></td><td></td><td>For 10% sucrose solution, if applicable</td></tr></tbody></table>

palatable western-style cafeteria diet as a reliable method for modeling diet-induced obesity in rodents

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;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.&#160;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.

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 ...

Watch this Scientific Journal Video about Palatable Western-style Cafeteria Diet as a Reliable Method for Modeling Diet-induced Obesity in Rodents at JoVE.com

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

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.

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;The health ...

palatable-western-style-cafeteria-diet-as-reliable-method-for

Research

JoVE Journal

Medicine

10.5K Views.  UNSW Sydney. 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.

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

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

Behavior

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 ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

Immunology and Infection

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits. Thanks to animal models, basic research can investigate the mechanisms underlying pathological processes such as MetS. Here, we describe the methods used to develop an experimental rabbit model of diet-induced MetS and its assessment. After a period of acclimation, animals are fed a high-fat (10% hydrogenated coconut oil and 5% lard), high-sucrose (15% sucrose dissolved in water) diet for 28 weeks. During this period, several experimental procedures were performed to evaluate the different components of MetS: morphological and blood pressure measurements, glucose tolerance determination, and the analysis of several plasma markers. At the end of the experimental period, animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia with low HDL, high LDL, and an increase of triglyceride (TG) levels, thus reproducing the main components of human MetS. This chronic model allows new perspectives for understanding the underlying mechanisms in the progression of the disease, the detection of preclinical and clinical markers that allow the identification of patients at risk, or even the testing of new therapeutic approaches for the treatment of this complex pathology.

We describe methods to develop an experimental model of diet-induced metabolic syndrome (MetS) in rabbits using a high-fat, high-sucrose diet. Animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia, thus reproducing the main components of human MetS. This chronic model will allow acquisition of knowledge underlying mechanisms of disease progression.

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased ...

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 ...

Vinyl Chloride and High-Fat Diet as a Model of Environment and Obesity Interaction

Vinyl chloride (VC), an abundant environmental contaminant, causes steatohepatitis at high levels, but is considered safe at lower levels. Although several studies have investigated the role of VC as a direct hepatotoxicant, the concept that VC modifies sensitivity of the liver to other factors, such as nonalcoholic fatty liver disease (NAFLD) caused by high-fat diet (HFD) is novel. This protocol describes an exposure paradigm to evaluate the effects of chronic, low-level exposure to VC. Mice are acclimated to low-fat or high-fat diet one week prior to the beginning of the inhalation exposure and remain on these diets throughout the experiment. Mice are exposed to VC (sub-OSHA level: &#60;1 ppm) or room air in inhalation chambers for 6 hours/day, 5 days/week, for up to 12 weeks. Animals are monitored weekly for body weight gain and food consumption. This model of VC exposure causes no overt liver injury with VC inhalation alone. However, the combination of VC and HFD significantly enhances liver disease. A technical advantage of this co-exposure model is the whole-body exposure, without restraint. Moreover, the conditions more closely resemble a very common human situation of a combined exposure to VC with underlying nonalcoholic fatty liver disease and therefore support the novel hypothesis that VC is an environmental risk factor for the development of liver damage as a complication of obesity (i.e., NAFLD). This work challenges the paradigm that the current exposure limits of VC (occupational and environmental) are safe. The use of this model can shed new light and concern on the risks of VC exposure. This model of toxicant-induced liver injury can be used for other volatile organic compounds and to study other interactions that may impact the liver and other organ systems.

The goal of this protocol was to develop a murine model of low-level toxicant exposure that does not cause overt liver injury but rather exacerbates pre-existing liver damage. This paradigm better recapitulates human exposure and the subtle changes that occur upon exposure to toxicant concentrations that are considered safe.

Vinyl chloride (VC), an abundant environmental contaminant, causes steatohepatitis at high levels, but is considered safe at lower levels. Although ...

Self-Administration of Drugs in Mouse Models of Feeding and Obesity

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of administration have significant effects on important metabolic parameters including food intake and body weight. Although an attractive option to circumvent this is to compound the drug in rodent food or dissolve it in water, these approaches also have limitations as they are affected by drug stability at room temperature for extended periods of time, the drug's solubility in water, and that the dosing is highly dependent on timing of food or water intake. The constant availability of the drug also limits translational relevance on how drugs are administered to patients. To overcome these limitations, drugs can be mixed with highly palatable food, such as peanut butter, allowing mice to self-administer compounds. Mice reliably and reproducibly consume the drug/peanut butter pellet in a short time frame. This approach facilitates a delivery approach with minimal stress compared with an injection or gavage. This protocol demonstrates the approach of drug preparation, animal acclimatization to placebo delivery, and drug delivery. The implications of this approach are discussed in studies related to timing of drug administration and the circadian rhythm.

The overall goal of this procedure is to describe a method for self-administration of drugs that can be used in mouse models of feeding and obesity.

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of ...

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.

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 ...

Biology

Murine Model for Diabetic Cardiomyopathy via High-Fat Diet and STZ Injection

Modeling and Evaluation of Murine Diabetic Cardiomyopathy Model

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus (T2DM), remain poorly understood. The myocardial toxicity associated with T2DM is attributed to factors such as lipotoxicity, glucotoxicity, oxidative stress, reduced cardiac efficiency, and lipoapoptosis. Compared to rats, mice offer greater accessibility, cost-effectiveness, and broader applicability for animal experiments. Insulin resistance and impaired insulin secretion are crucial factors in the pathophysiology of T2DM. We introduce a novel nongenetic murine model that replicates the progression of human DbCM induced by a combination of high-fat diet (HFD) feeding and streptozotocin (STZ) injection. In this study, we used wild-type C57BL/6J mice, administering an HFD regimen for 12 weeks, followed by intraperitoneal injections of STZ for an additional 12 weeks to induce characteristic manifestations of T2DM. We conducted oral glucose tolerance tests and measured serum insulin concentrations to confirm the development of insulin resistance and insufficient insulin secretion. Cardiac structure and function were rigorously assessed through noninvasive transthoracic echocardiography. Pathological characteristics were evaluated through Masson's trichrome staining and wheat germ agglutinin (WGA) staining, revealing pathological features related to DbCM. Therefore, we provide a robust and versatile method for establishing a nongenetic murine model of DbCM.

This protocol outlines a method for inducing diabetic cardiomyopathy through a combination of high-fat diet feeding and streptozotocin injection. This approach aims to provide a reliable framework for scientific investigation into diabetic cardiomyopathy and to explore potential avenues for clinical treatment applications.

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus ...

Manipulation of Rhythmic Food Intake in Mice Using a Custom-Made Feeding System

Rhythmic gene expression is a hallmark of the circadian rhythm and is essential for driving the rhythmicity of biological functions at the appropriate time of day. Studies over the last few decades have shown that rhythmic food intake (i.e., the time at which organisms eat food during the 24 h day), significantly contributes to the rhythmic regulation of gene expression in various organs and tissues throughout the body. The effects of rhythmic food intake on health and physiology have been widely studied ever since and have revealed that restricting food intake for 8 h during the active phase attenuates metabolic diseases arising from a variety of obesogenic diets. These studies often require the use of controlled methods for timing the delivery of food to animals. This manuscript describes the design and use of a low-cost and efficient system, built in-house for measuring daily food consumption as well as manipulating rhythmic food intake in mice. This system entails the use of affordable raw materials to build cages suited for food delivery, following a user-friendly handling procedure. This system can be used efficiently to feed mice on different feeding regimens such as ad libitum, time-restricted, or arrhythmic schedules, and can incorporate a high-fat diet to study its effect on behavior, physiology, and obesity. A description of how wild-type (WT) mice adapt to the different feeding regimens is provided.

Restricting the timing of food intake has emerged as a promising intervention to attenuate diet-induced metabolic diseases. This manuscript details the construction and use of an efficient system built in-house for measuring and manipulating rhythmic food intake in mice.

Rhythmic gene expression is a hallmark of the circadian rhythm and is essential for driving the rhythmicity of biological functions at the appropriate ...