WUSTL Diabetes Models Phenotyping Core
Diabetes is a systemic disease, affecting every organ in the body. In vitro and cell culture approaches can provide important molecular insights into diabetes, and human studies can provide key observational clues about diabetes, but well-defined physiological models of diabetes are essential for bridging molecular understanding to human pathophysiology. The Diabetes Models Phenotyping Core provides state-of-the-art services to multiple funded research projects in the area of diabetes and its complications so that rigorous experiments performed in model systems have the potential to yield compelling results that can be translated to a clinical context.
Biochemical Analysis of Serum
Standardized assays of mouse serum include glucose, non-esterified fatty acids, triglycerides, and cholesterol, which have been adapted for high throughput colorimetric assays performed in very small volumes. Transaminases are also quantified. In addition, the Core assays mouse lipoproteins, a more labor-intensive service that requires separation of plasma by size exclusion chromatography.
Glucose Tolerance and Insulin Tolerance Testing in Mice
For glucose tolerance testing, dextrose is administered to fasted mice (1 mg/g body weight i.p.), and for insulin tolerance testing, human insulin is administered (0.75 units/kg body weight). Whole blood from the tail is analyzed over time for blood glucose. Two available options include 1) user-based approach after education and training by Core personnel, and 2) testing in a Core facility (Room 845 of the Southwest Tower) by an experienced technician. Option 1 enables testing to be performed where the mice are initially housed and decreases variability due to stress of animal transfers.
Determination of Body Composition
In rodents, this is accomplished using an EchoMRI device for those scientists interested in quantifying only adiposity and lean body mass. The core also provide access to determinations using a DEXA machine, which overestimates fat mass but has considerable precision for mineral determinations.
Assaying Metabolic Parameters including Metabolic rate and Food intake
Comprehensive metabolic, behavioral, and physiological analyses are provided using the TSE/PhenoMaster an automated metabolic caging system that measures oxygen consumption, carbon dioxide generation, physical activity, in addition to food and liquid intake. An Oxymax system is also available to measure oxygen consumption by indirect calorimetry. The core also provides independent analysis of food intake and water intake as well as mouse excretions in metabolic cages.
Blood Pressure Determinations
We offer both noninvasive tail cuff evaluations as well as invasive telemetry-based evaluations. Our tail cuff system is the Columbus Instruments’ Non-Invasive Blood Pressure Monitor system (NIBP S/N 110707-1), which can determine systolic, diastolic and mean BP in up to eight mice or rats. Animals are subjected to an acclimatization protocol and are studied using a warming restrainer as tail vessels constrict in response to cold. The telemetry system involves surgical placement of telemetry catheters that provide information regarding blood pressure as well as temperature and physical activity. Data are recorded after animals appropriately recover from surgery.
Quantitative Atherosclerosis Analysis
Dissection microscopes, aortic processing equipment, computerized image capture systems for quantifying lesions, and instruction in design of experiments as well as isolation and pinning of mouse aortas are provided.
Biochemical Analyses of Tissues.
Utilizing specific mouse organs (heart, liver, skeletal muscle, etc.) or whole bodies of model organisms such as Drosophila, lipids are extracted using protocols with optimized organic solvent mixtures (e.g., chloroform/methanol), the solvent is dried under nitrogen, the lipid is appropriately resuspended in an assay mixture, and triglycerides, cholesterol, and phospholipids are quantified using enzymatic techniques that are validated in each assay run with the use of appropriate positive and negative controls as well as standard curves. When warranted, internal standards can be included to monitor extraction efficiency. These results are normalized to either tissue weight or protein content, the latter determined by assaying an aliquot of the tissue prior to the lipid extraction.
Training for Breeding and Genotyping
The core provides instruction for establishing and maintaining mouse colonies, including breeding strategies to maximize yield, optimal weaning ages, identifying unsuitable dams, simple mouse anesthesia, genotyping techniques, mouse identification techniques, mouse handling and blood drawing, determination of pup sex, basic Mendelian inheritance, and for certain users, basic animal surgery techniques encompassing catheter and pump placement, sterile technique, and post-operative analgesia.
Provision of Diabetes Models
The Core now provides models that in the proper context could be viewed as relevant to type 1 diabetes.
- Non-obese Diabetic (NOD) mice (NOD/ShiLtJ). The incidence of immune-mediated diabetes for colonies maintained in our animal facilities is nearly 90% for female mice between the age of 20 and 30 weeks.
- Akita mice. Heterozygotes with the Ins2C96Y allele (in the C57BL/6 background) develop beta-cell failure due to endoplasmic reticulum stress and diabetes in the setting of insulin deficiency in the absence of obesity. Unlike the NOD model, this phenotype is more robust in males.
The Core now provides two models that in the proper context could be viewed as relevant to type 2 diabetes.
- db/db mice (C57BLKS background). Homozygotes at the Leprdb locus develop diabetes, obesity, and hyperinsulinemia.
- C57BL/6 diet-induced obesity (DIO) mice.Mice are supplied following 3 weeks of high fat diet (Harlan Teklad TD88137: 42% kcal fat, 43% kcal carbohydrate, 15% kcal protein, beginning at 6 to 8 weeks of age) to induce insulin resistance or following 6 weeks on high fat diet to induce hyperglycemia and body composition changes. Alternatively, mice are supplied on standard breeding chow (PicoLab 5053: 13% kcal fat, 62% kcal carbohydrate, 25% kcal protein) and investigators counseled regarding diet, expected phenotypes, and optimal ages for interventions.
In Vivo Spectral Imaging
The Core provides imaging instrumentation for studying diabetes-relevant variables that can be visualized in living animals using a Kodak Multispectral Imaging System, which is suitable for applications that include reporter mice (using luciferase), fluorescently labeled antibodies and other molecules of various sizes, and high resolution imaging of the vasculature using contrast agents.