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

Abstract

Background and Purpose: Glucose tolerance tests (GTTs) are commonly used in preclinical studies but are poorly standardised. Male mice are often preferred due to more severe phenotypes which may aid in detecting drug effects. Using novel glucose telemetry in undisturbed mice, the effect of different pre-GTT protocols on blood glucose concentrations, GTTs and detection of drug effects were considered. Experimental Approach: Seven male and female C57Bl/6J mice (8-10 weeks) were implanted with HD-XG glucose telemetry devices. Mice were fasted for 16h overnight or 6h in the daytime following a whole cage change, cage change with retention of used bedding or no cage change prior to i.p.GTTs. Glucose tolerance following oral glucose gavage was compared to voluntary ingestion of gels. Using the most refined procedures, 250mg/kg oral metformin and 10nmol/kg i.p. exendin-4 were tested. Key Results: Blood glucose initially increased following cage changing at the start of the fast. For 6h fasting, retaining bedding reduced these initial responses and produced more timely glucose reductions whereas 16h fasts caused pronounced hypoglycaemia. Impaired glucose tolerance in males was exaggerated following 16h fasting or whole cage changes. Refined procedures including voluntarily ingested glucose gels blunted responses but the effects of exendin-4 and metformin were still observable in both sexes. Conclusion and Implications: Variations in GTT protocol can have profound effects on glucose homeostasis. Improved glucose tolerance due to protocol refinement and/or the use of females still allows for detection of drug effects providing evidence that more severe phenotypes are not required when testing drugs.

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

“…As with overnight fasting, cage change method not only impacts effectiveness of the fast but also GTT outcome with whole cage changes at the start of a 6-h fast significantly impairing glucose tolerance during an i.p.GTT when compared with when bedding is retained or the cage is not changed. 24 Overall, these results highlight the role of welfare in scientific outcomes and the importance of consistency in practices and full disclosure of methods. 4.1.2 | Moving animals from holding rooms to procedure rooms In many animal units, mice are transported from holding rooms to procedure rooms prior to experimentation (e.g., GTTs).…”

“…16,17,23 Furthermore, cages are sometimes changed at the start of fasting to eliminate the risk of food remnants being left in the cage. 24,25 However, whether and/ or how this is done is not consistent or well documented with the potential impacts of this discussed later in this review.…”

“…16 Data from our own laboratory using CGM also indicate that 16-h fasts induce severe hypoglycaemia with blood glucose concentrations consistently falling <2.8 mM at ~13 h after the start of fasting. 24 More recently, it has become more common practice to fast for shorter periods (3-6 h) during the daytime and this has been shown to be sufficient for ensuring gastric emptying and hepatic control of glucose homeostasis. 18,[35][36][37] However, overnight fasting still features in many research papers.…”

“…Interestingly, overnight fasts not only have welfare implications but can significantly alter glucose response during an i.p.GTT with 16-h fasts being associated with impaired glucose tolerance particularly in males. 24 Another poorly reported variable in the fasting protocol is whether the cage is changed at the start of the fast. Although no food remnants will remain if the cage is changed, this procedure is associated with stress responses in mice, including increased corticosterone, most likely due to removal of familiar odours.…”

“…38 Indeed, our data have shown that bedding retention reduces the initial glucose spike associated with researcher intervention at the start of the fast. 24 While removing food from the food hopper without changing the cage causes the least amount of stress at the start of the fast, the blood glucose concentration reductions are not as significant, most likely due to food remnants present in the bottom of the cage.…”