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.