Introduction
Obesity has reached pandemic levels in Western society, contributing to increased healthcare burdens and decreased life expectancy. As a complex, chronic disease involving decades of pathophysiological changes, determining the exact mechanisms in humans is challenging. To circumvent these issues, researchers utilize surrogate models, including murine genetic mutations, transgenic mutations, polygenic models, and environmental exposure models. The diet-induced obesity (DIO) model has become a crucial tool for understanding the interplay between high-fat Western diets and the development of obesity, closely mimicking the increasing availability of high-fat/high-density foods in modern society.
The Diet-Induced Obesity (DIO) Model
The Diet-Induced Obese (DIO) mouse model is a gold standard for studying obesity and metabolic disorders in preclinical research. Developed on the C57BL/6NTac background, this model reliably develops obesity, insulin resistance, and other metabolic phenotypes when fed a high-fat diet. Its predictable response to dietary manipulation makes it an essential tool for researchers investigating the mechanisms of obesity, type 2 diabetes, and related metabolic diseases.
Mimicking Human Obesity
The DIO model closely mimics the increasing availability of high-fat/high-density foods in modern society over the past two decades, which are main contributors to the obesity trend in human. This model has lead to many discoveries of the important signalings in obesity, such as Akt and mTOR.
Key Considerations for DIO Models
Several factors are important when using this model of diet-induced obesity. First, the genetic nature and degree of model characterization as well as differences in mouse background could lead to different phenotype. For examples, AKR/J and DBA/2J mice are very responsive to high-fat diet, while A/J and Balb/cJ mice are more resistant. C57BL/6J mouse are better characterized than some of the other models. Second, gender usually plays an important role. Generally, male mice are more affected by diabetes than are female mice and thus are used more often in diet-induced obesity studies. Third, environmental factors are important and should be considered. Obese mice are sensitive to stress and their environmental settings will affect experimental results. Therefore, cage placement, mice density, food quality, mice handling, beddings and mice-check frequency will all results in disturbance of the development of obesity in experimental mice.
The C57BL/6 Mouse Model
The C57BL/6J mouse model has been shown to be a particularly good model mimicking human metabolic disorders that are observed in obesity. When fed ad libitum with a high-fat diet (HFD), obesity, hyperinsulinemia, hyperglycemia and hypertension establish in these mice. Control groups fed ad libitum with normal chow however, do not develop any metabolic abnormalities.
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Genetic Uniformity and Reliability
Black 6 (C57BL/6) DIO mice are the preferred choice for many researchers because of their proven track record in various research applications. These genetically uniform mice ensure that your experiments are not affected by genetic variability, allowing for clear and reliable data. C57BL/6 (B6) DIO mice are the trusted choice for researchers seeking consistency and reliability in preclinical studies.
Metabolic Abnormalities
The metabolic abnormalities of B6 mouse closely parallel that of human obesity progression pattern. Although an increase in body weight can be noticed after 2 weeks, the increase is gradual and becomes apparent after 4 weeks. After 16-20 weeks of high-fat diet feeding, mouse will typically exhibit 20-30% increase in body weight when compared to chow-fed mouse. The high-fat diet's effects on blood glucose are more discrepant and depend on the type of dietary regimen. Hyperglycemia usually develops within 4 weeks of a high-fat diet. The elevation of fasting glucose is usually accompanied by increases in fasting insulin levels. At that time, hyperinsulinemic-euglycemic clamp experiments will demonstrate whole-body insulin resistance. However, there are no reliable predictors for diabetes development or onset and the reported development of overt diabetes is controversial.
Akt and mTOR Signaling
Akt and mTOR pathyway integrates several important signals that regulate cell growth and metabolism. Activation of Akt and mTOR signalings by food intake has great implications in obesity and insulin resistance. Inhibition of Akt and mTOR pathway by rapamycin has effects in longevity, adipocyte differentiation, and obesity.
Taconic Biosciences and the DIO Model
When it comes to B6 DIO mice, Taconic Biosciences is your trusted partner. With decades of experience in breeding and maintaining high-quality mouse models, Taconic ensures that every Black 6 (C57BL/6) DIO mouse meets the strictest standards for genetic purity and health. Taconic Biosciences offers B6 mice of exceptional quality, ready to support your next study. To help alleviate uncertainty in selecting a new model, we offer a free animal trial program that allows you to test the quality, consistency, and availability of Taconic’s rodent models.
Diet and Housing Conditions
Taconic Biosciences maintains an inventory of C57BL/6NTac male mice on 60% kcal high fat irradiated diet. Taconic uses Research Diets industry standard high fat rodent diet, D12492. C57BL/6NTac males are put on special diet at 6 weeks of age and housed at reduced density. Control males are housed in the same location, also at reduced density, and are fed NIH-31M diet. Animal Diet: DIO-B6-M are fed the D12942 diet and B6-DIOCONTROL-M are fed the NIH #31M Rodent Diet. If a low-fat purified ingredient diet is preferred, recommended control diets include D12450J and D12450K from Research Diets, though other variations are possible.
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Mitigating the Impact of Extended Transit
DIO mice in transit for an extended period of time (more than 48 hours) can lose 20-25% of their body weight and reach a new weight set point, making them resistant to high fat diet when they arrive. To help mitigate the impact of extended transit on DIO phenotype stability, Taconic maintains regionally available cohorts of DIO mice.
Nnt Gene Mutation
The NTac substrain does not carry the spontaneous mutation in the nicotinamide nucleotide transhydrogenase (Nnt) gene, which is present in other B6 substrains and known to affect glucose metabolism.
Ordering Specifications
DIO mice are placed on a high fat diet (D12492) starting at 6 weeks and are packed within a 10 gram maximum weight span. Controls are fed NIH-31M. We are not able to take DIO orders by weight, but can assist in selecting an appropriate age to meet you weight requirement. All DIO mice will meet the above specifications at the time of packing. Use the Health Standard Selector to enter your exclusion list.
Metabolic Parameters in DIO B6 Mice
Insulin, fasted blood glucose and HOMA-IR in DIO B6 compared to wild type C57BL/6NTac controls (A-C) or for DIO B6 at two different longitudinal timepoints (D-F). Insulin sensitivity assessed after an acclimation period. Animals were fasted for 5-6 h. After fast, blood glucose levels taken via tail nick with One Touch glucometer. Immediately, extra blood collected for insulin ELISA. Blood was collected in EDTA-coated microvette tubes (Fisher # NC9041302). Ultra Sensitive Mouse Insulin Kit from Crystal Chem (#90080) used to perform insulin ELISA, with protocol followed according to manufacturer's instructions.
Semaglutide Treatment
Metabolic parameters in Taconic's Diet Induced Obese (DIO) B6 mouse in response to twice weekly subcutaneous injection of semaglutide (30 nmol/kg) or vehicle (N = 9/group). Mice were diet conditioned for 12 weeks prior to shipment to the study site and were acclimated 1.5 weeks prior to study initiation. Body weight change (A) and food intake (B) were reduced by semaglutide treatment. Active GLP-1, glucagon, and insulin were measured 4 hours after final treatments were administered (C-E). Active GLP-1 was significantly elevated compared to vehicle in the semaglutide group, while glucagon and insulin were significantly lowered compared to vehicle.
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Diets for Inducing Obesity
Diverse high energy diets have been utilized to induce obesity and related metabolic disorders in rodent models, though the dietary mediation has not been absolutely standardized. Traditionally, these diets consist of a simple exchange of carbohydrate-derived calories with fat-derived calories and are compared to a standard chow diet (SCD) as control. Though, this poses the problem that HFD compared to the control exhibits marked differences in micro- and macronutrient content. Therefore, obtained results cannot be accurately attributed to HFD only.
High-Fat Diet Composition
Purified high fat diets used to induce obesity and obesity-related complications such as diabetes and metabolic syndrome typically have 40-60% of energy derived from fat. Diets with 55-60% of calories from fat like TD.06414 and TD.93075 are commonly used for inducing obesity in rodents. While considered extreme compared to typical human fat consumption, these diets are effective in initiating rapid weight gain in most rodents. With higher fat content there is less room for carbohydrate, thus the carbohydrate (particularly sucrose) amount is relatively low compared to other obesity inducing diets. As the fat level increases, pellet quality (durability) is often compromised. Some higher fat formulas are available only in non-pelleted form or require specific carbohydrate, maltodextrin, for pelleting. Depending on the fat and carbohydrate sources used, the non-pelleted form could be dense and crumbly, dough-like, or paste-like.
"Western Diets"
Diets with 40-45% of calories from fat, like TD.95217 , TD.88137 , TD.06415 , and TD.08811 , represent another popular diet pattern for diet-induced obesity work. These diets have double or triple the amount of sucrose found in higher fat diets. High levels of simple carbohydrate like sucrose and fructose may help to promote hypertriglyceridemia, insulin resistance, and fatty liver. Diets with a pattern of high sucrose and high saturated or trans fat are often referred to as “Western Diets” in obesity and cardiovascular fields. Some “Western Diets” have further modifications to the fatty acid profile or even specific vitamin and minerals adjustments to be even more closely matched to a Western Diet pattern.
Control Diets
Many researchers choose to compare their high fat fed animals to animals fed a natural ingredient, grain-based diet (also referred to as standard diets or chow). These diets differ in the source and level of nutrients as well as in the presence of non-nutritive factors (such as phytates or phytoestrogens). Depending on what your main comparisons are, it may be suitable to have a grain-based diet as your control/reference group.
A very basic purified control diet would be AIN-93M (TD.94048 ) or AIN-93G (TD.94045 ). AIN-93 diets have a moderate amount of sucrose at ~10%, and fat is from soybean oil with a healthy fatty acid profile.
Experimental Procedures in DIO Models
Diet induced obesity model usually takes more than 16-20 weeks for completing the experiments. Moreover, obesity is a multiple organ system disease, involving derangements of bodyweight, adipose tissue, glucose metabolism, cholesterol level, and inflammatory markers. Therefore, preexperimental design is crucial for the success of the experiments. While multiple organ systems could be tested and many parameters could be examined, it is important to have upfront experimental hypothesis and goals. During obesity development, one could examine and measure a wide variety of parameters. However, different hypotheses will required different measurements and experimental methods, which in some cases, could affect physiologic parameter.
Animal Care and Maintenance
- Keep mice under standard pathogen-free conditions with food and water ad libitum and regular 12:12 light-dark cycle.
- Check cage and bedding conditions daily but avoid check mice too frequently.
- Pay attention to the environment nearby the mice cages.
Baseline Data Collection
As there is also individual variation even in the inbred strains of the mice, it is important to collect the baseline data of the mice before experiments. We measure body weight, tibial length, fasting glucose for mice of the same gender and age. The baseline fasting glucose of C57BL/6J male mice ranges from 40-190 mg/dl at age of 6 weeks in our laboratory; we only recruit mice with their fasting glucose between 70 and 130 mg/dl. We also exclude the male mice with body weight below 15 g or over 25 g at 6 weeks of age.
- Measure baseline glucose: insert glucometer strip into glucometer.
- Pick up mouse with gloves.
- Restrain the mice with either the other hand or the mice restrainer. We usually use unheated version of rodent restrainer. It s cylindrical and have series of holes on each side to facilitate injections or blood testing.
Diet Regimen and Food Intake
- Select the different diet regimen.
- Measure the diet weight before change the diet.
- Pour the diet into the cages and avoid pouring into the fragments or powder into the cages as they sometimes contaminated the beddings with polyuric mice.
- Observe daily for the food color. These 60% high-fat diet tends to become moisture and oily after 2-3 days exposing to air.
- Change the diet every week and measure the changes in food weight for measuring the food intake of the mice. Usually put more food as needed. We do not add fresh food between each measurement as it could result in differences between groups.
- When obese mice develop polyuria, it may require changing bedding more than two to three times per week.
Blood Collection
- Pick up mouse with gloves.
- Restrain the mice with either the other hand or the mice restrainer. We usually use unheated version of rodent restrainer. It s cylindrical and have series of holes on each side to facilitate injections or blood testing.
- For serum or more blood other than glucose, use capillary tube.
Glucose Tolerance Test (GTT)
Accurate glucose tolerance test depends on the preparation of the mice. To closely mimic the human part of the glucose tolerance test, we perform mice fasting from 7:00 AM to 15:00 PM. Mice are active during the light-dark phase and this fasting period are more physiological when comparing to human. In our laboratory, we usually administer glucose by intraperitoneal injection. Injection is more consistent with dosage and the results. It also saves time if many mice are going to be tested.
- Clean the bedding and the food from the cage. Make sure that there is no food left in the beddings.
- Prepare a sterile 10% (w/v) glucose solution in PBS. Glucose will be administered at a concentration of 1 g/kg (glucose/ body weight) in a 10 ml/kg volume. Occasionally, we choose 2 g/kg concentration earlier in the experiments before mice become diabetic.
- Preload 1 ml insulin syringes with the glucose solution.
- After fasting period is complete. Measure baseline glucose level.
- Pick up mouse with gloves.
- Restrain the mice with either the other hand or the mice restrainer.
- Cut the tail as less as possible with scalpel.
- Put one drop of the blood from tail onto glucometer strip and reads. Record glucose value. The range of glucose values is 20-600 mg/dl. If the glucometer indicates the value “Hi”, we usually take the blood with capillary and diluted 2× with PBS.
- After adequate fasting period, inject mice with intraperitoneal glucose.
Insulin ELISA
- After serum collection, store the samples at -20°C.
- Prepare reagent. Dilute the conjugate with ten parts of conjugate buffer.
- Reconstitute insulin control with 0.6 ml distilled water.
- Ensure that microplates are at room temperature prior to opening foil pouch.
- Add 75 μl of enzyme conjugate into each well.
- Wash the microplate for six times with wash buffer.
- Pipette 100 μl of stop solution into each well.
- Read the absorbance at 450 nm with a reference wavelength of 620-650 nm.
Glucose-Stimulated Insulin Secretion Test
We usually perform glucose-stimulated insulin secretion test alone with glucose tolerance test. The peak insulin secretion after glucose intraperitoneal injection is around 9-10 min. We typically collected sample at 0, 5, 10, 15, and 30 min after glucose injection. Spin the capillary tubes into a microhematocrit centrifuge at 12,000 g for 10 min. Add the serum into the microplates.
Insulin Tolerance Test (ITT)
The dose of insulin tolerance test may vary from 1 to 5 U/kg and should take into consideration of the body weight, diabetic status and genetic background of the mice. In the C57BL/6J diet induced obesity model, we usually use 2 U/kg insulin for injection. We usually perform insulin tolerance test in fasting condition.
- Prepare insulin and glucose. Dilute insulin into 2 U/10 ml by iced sterile PBS.
- Preload the injection syringes with insulin and keep them on ice.
- Measure glucose level and observe the mice.
Body Composition Analysis
With the advances of the technology, magnetic resonance imaging (MRI) and dual energy X-ray absorptiometry scanning (DEXA) has gradually replaced the chemical carcass analysis for measuring the fat component. Chemical carcass used to be considered as the gold standard for whole body composition analysis. It requires to sacrifice the mice and time-consuming. Dissecting and weighing of fat tissues in individual mice can also measure the total body fat. However, this method is less accurate. Body composition measurements by DEXA consist of three components: fat, bone mineral, and lean soft tissue. It shows strong correlation between the percentage of fat area and total body fat as compared with chemical extraction method.
Tissue Collection and Analysis
After the diet feeding experiments, mice are sacrifice to collect their blood, internal organ and analyze the whole body composition. Diet induced obesity model will result in multiple organ changes when comparing to its control mice. High-fat diet will lead to hepatic steatosis and fatty liver changes could be obviously observed after dissection. Adipocyte number and size are increased. Pancreatic islets will become hypertrophic and then degenerate. Skeletal muscle may have decreased insulin sensitivity. Brown fat may show atrophy or white fat deposition. Hence, obesity is a whole body system derangement.
- Mark every eppendorf tubes for serum ample, tissue collection or histological fixation.
- Insert a needle at the left ventricle apex of the heart and draw the blood slowly. Avoid drawing the blood too soon.
- Proceed with histology or tissue analysis
Limitations of the DIO Model
Obesity is affected by "environmental, biological, and psychosocial pressures," therefore it is understandable that several limitations are established when translating results between the results of a diet induced obesity model in a lab and humans. Numerous sources of biological variation arise in rodents before translating results to humans is even considered. Furthermore, the strain and sex of the rodent impacts the response to the model. With varied sources and types of fat, researches have shown the complexity of defining a model of a high fat diet that can both resemble human meals and accurately quantify the nutrient contents. There is significant variation in the results appearing in either experimental rodents that were fed with high fat diets made up from different ingredients and from purified ingredient. Additionally, they both can be found in rodents' and humans' diets. Researchers have created the study models of high carbohydrate and high proteins. However, the variation in the results of those models has caused the difficulties to interpret and find the relation to human case. Similar to fat (lipid), the sources of protein and carbohydrate are also essential contributors to the outcomes of high fat diets and control diets rodents group. Given the diversity in human food and each human individually distinguished metabolic capacity, the results of testing the diet induce obesity in rodents are limited in term of translatability. Laboratories temperature which are usually (18-22 °C) are lower than the thermoneutrality of model organisms like mice which are about 30 °C. That can introduce hyperphagia in the organism in effort of increasing their metabolism to generate heat energy for the body.
Rodents are nocturnal and are mostly feeding at night, in their natural habitat. The changes in the light/dark cycle of laboratories can alter their circadian rhythm which can affect their metabolism. Besides that several studies used genetically modified mouse models that have decreased circadian rhythmicity gene. The cause of type 2 diabetes mellitus in humans are far more complicated than the sole consumption of a high fat diet. The mental, emotional and cultural factors along with insulin resistance and hyperphagia are known to increase the occurrence of type 2 diabetes in humans. However, type 2 diabetes in model organisms are introduced via surgery of partial or whole pancreas, or using chemicals such as streptozotocin. Streptozotocin inhibits the ability of pancreatic β cells to produce insulin, and depending on the dosage used, the result can be partial or absolute inhibition.
DIO Models in Cancer Research
In vivo animal models are important research tools to study the underlying mechanisms of the association between obesity and cancers. Among genetic models of obesity, mice deficient in leptin signaling are the most used. When mice were fed standard chow, the genetic model showed early-onset obesity and comorbid diseases such as insulin resistance and hepatic steatosis. Their main disadvantage is the exclusion of the factors other than leptin that may affect cancer cells and tumor microenvironment. For example, obesity accelerates the progress of Kras-driven pancreatic ductal adenocarcinoma, but not lung cancer.
The DIO mouse model is believed to mimic human obesity well and to explain the potential biological link between obesity and cancer. The DIO model was established by feeding mice a diet high in sugar, fat or both. While several feeding regimens have been developed, the most commonly used diets contain 30% to 60% kcal from fat, which is fed to the mice for 10 to 12 weeks prior to tumor formation.
Most obesity-related complications are due to inflammation. Chronic inflammation in adipose tissue, especially white adipose tissue (WAT), stimulates cancer progression through mechanisms such as altered levels of adipokines and inflammatory mediators, and insulin resistance. Short-term HFD feeding is difficult to obtain an ideal model sufficient to study the relationship between obesity and cancer. Therefore, long-term obesity models need to be established to simulate the relationship between human obesity and tumors. The feeding time of the HFD-induced obesity mouse model ranged from 4 weeks to 56 weeks, and 10 weeks to 12 weeks were usually selected. DIO mice gain weight, increase fasting blood glucose levels, and develop obesity-related phenotypes such as hyperinsulinemia, insulin resistance, hepatic steatosis, hypertension, and dyslipidemia. Whether reversal of the obesity phenotype affects tumor prognosis is a key question in this field.
Nude mice used to establish tumor xenograft models, such as BALB/c, are generally difficult to induce obesity. Stemmer K et al. found that Foxn1 nude mice (B6. Cg-Foxn1nu/J) on a C57BL/6 background fed a high-fat diet under thermoneutral (33°C) conditions significantly increased their body weight, making them an excellent model for studying obesity and tumors.