The glucose tolerance test in mice: Sex, drugs and protocol.

AIM: To establish the impact of sex, dosing route, fasting duration and acute habituation stress on glucose tolerance test (GTT) measurements used in the preclinical evaluation of potential glucose-modulating therapeutics. METHODS: Adult male and female C57Bl/6J mice, implanted with HD-XG glucose telemetry devices, were fasted for 16 hours or 6 hours following acute habituation stress due to whole cage change, cage change with retention of used bedding or no cage change prior to intraperitoneal (IP) GTTs. To evaluate protocol refinement and sex on the ability of the GTT to detect drug effects, we administered 250 mg/kg oral metformin or 10 nmol/kg IP exendin-4 using optimized protocols. RESULTS: Female mice were less sensitive to human intervention when initiating fasting. Following a 6-hour fast, retention of bedding whilst changing the cage base promotes quicker stabilization of basal blood glucose in both sexes. Prolonged fasting for 16 hours resulted in an exaggerated GTT response but induced pronounced basal hypoglycaemia. Following GTT protocol optimization the effect of exendin-4 and metformin was equivalent in both sexes, with females showing a more modest but more reproducible GTT response. CONCLUSIONS: Variations in GTT protocol have profound effects on glucose homeostasis. Protocol refinement and/or the use of females still allows for detection of drug effects, providing evidence that more severe phenotypes are not an essential prerequisite when characterizing/validating new drugs.

The use of mice in diabetes research: The impact of experimental protocols.

Mice are used extensively in preclinical diabetes research to model various aspects of blood glucose homeostasis. Careful experimental design is vital for maximising welfare and improving reproducibility of data. Alongside decisions regarding physiological characteristics of the animal cohort (e.g., sex, strain and age), experimental protocols must also be carefully considered. This includes choosing relevant end points of interest and understanding what information they can provide and what their limitations are. Details of experimental protocols must, therefore, be carefully planned during the experimental design stage, especially considering the impact of researcher interventions on preclinical end points. Indeed, in line with the 3Rs of animal research, experiments should be refined where possible to maximise welfare. The role of welfare may be particularly pertinent in preclinical diabetes research as blood glucose concentrations are directly altered by physiological stress responses. Despite the potential impact of variations in experimental protocols, there is distinct lack of standardisation and consistency throughout the literature with regards to several experimental procedures including fasting, cage changing and glucose tolerance test protocol. This review firstly highlights practical considerations with regard to the choice of end points in preclinical diabetes research and the potential for novel technologies such as continuous glucose monitoring and glucose clamping techniques to improve data resolution. The potential influence of differing experimental protocols and in vivo procedures on both welfare and experimental outcomes is then discussed with focus on standardisation, consistency and full disclosure of methods.

The use of mice in diabetes research: The impact of physiological characteristics, choice of model and husbandry practices.

Diabetes mellitus is characterised by hyperglycaemia, which results from an absolute or relative lack of insulin. Chronic and acute hyperglycaemia are associated with a range of health complications and an overall increased risk of mortality. Mouse models are vital in understanding the pathogenesis of this disease and its complications, as well as for developing new diabetes therapeutics. However, for experimental questions to be suitably tested, it is critical that factors inherent to the animal model are considered, as these can have profound impacts on experimental outcome, data reproducibility and robustness. In this review, we discuss key considerations relating to model choice, physiological characteristics (such as age, sex and genetic background) and husbandry practices and explore the impact of these on common experimental readouts used in preclinical diabetes research.

Practical Considerations when Using Mouse Models of Diabetes.

Mouse models of diabetes are important tools used in preclinical diabetes research. However, when working with these models, it is important to consider factors that could influence experimental outcome. This is particularly important given the wide variety of models available, each with specific characteristics that could be influenced by extrinsic or intrinsic factors. Blood glucose concentrations, a commonly used and valid endpoint in these models, are particularly susceptible to manipulation by these factors. These include potential effects of intrinsic factors such as strain, sex, and age and extrinsic factors such as husbandry practices and experimental protocols. These variables should therefore be taken into consideration when the model is chosen and the experiments are designed. This chapter outlines common variables that can impact the phenotype of a model, as well as describes the methods used for assessing onset of diabetes and monitoring diabetic mice.

Continuous Glucose Monitoring in Conscious Unrestrained Mice.

Measurement of blood glucose concentration is a common end point in studies using animal models of diabetes. Usually a blood glucose meter is used to measure non-fasted blood glucose concentrations, typically at frequencies of between 1 and 7 times per week. This process involves pricking the tip of the tail to collect a small blood sample (0.5-5 µL), which could potentially cause a stress response and affect blood glucose concentrations. Moreover, with blood glucose concentrations constantly fluctuating in response to feeding and activity, a single-point measurement can easily misrepresent the actual glycemic control of the animal. In this chapter, we discuss the use of continuous glucose monitoring in mice by radio-telemetry which allows second-by-second changes in blood glucose to be captured without restraining the mouse. Glucose excursions rather than single-point measurements may prove more useful in detecting effects of treatments, and lack of handling may avoid stress responses causing artefacts. We outline what is involved in implanting such devices into mice including some practical tips to maximize success.