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

Abstract

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.

Keywords: animal; antidiabetic drug; continuous glucose monitoring (CGM); mouse model; pharmacology; type 2 diabetes.

FIGURE 1

The initial effects of 6‐hour and 16‐hour fasting on blood glucose concentrations from 30 minutes pre‐intervention to 120 minutes post‐intervention in (A) males, (B) females in proestrous‐oestrous (P‐E) and (C) females in metoestrous‐dioestrous (M‐D). (D) Area under the curve (AUC)₁₂₀ for graphs (A‐C). (E) Difference between maximum blood glucose reached from 0 to 120 minutes following intervention and pre‐intervention baseline. (F) Time spent above pre‐intervention blood glucose concentrations from 0 to 120 minutes post‐intervention. Pre‐intervention glucose was quantified as the average glucose between −30 and −15 minutes. * and # represent a significant difference compared to females in P‐E and M‐D, respectively. ~, +, = and ⱡ represent a significant difference compared to 6 hours with whole cage change (WCC), 6 hours with bedding retention cage change (BRCC), 6 hours with no cage change (NCC) and 16 hours with BRCC, respectively (P < 0.05, two‐way ANOVA with Holm‐Sidak post hoc tests). Data are mean ± SEM (n = 7 for males, n = 5‐7 for females in P‐E and n = 4‐7 for females in M‐D)

FIGURE 2

The prolonged effects of 6‐hour (6h) and 16‐hour (16h) fasting on blood glucose concentrations from 30 minutes pre‐intervention to 330 minutes and 960 minutes post‐intervention for 6‐hour and 16‐hour fasts, respectively, in (A) males, (B) females in proestrous‐oestrous (P‐E) and (C) females in metoestrous‐dioestrous (M‐D). (D) Time after start of fast that blood glucose fell below pre‐intervention concentrations for 5 consecutive minutes. (E) Minimum blood glucose concentration reached during fasting compared to normal 24‐hour minimum glucose concentrations with ad libitum food. (F) Percentage of time spent below pre‐intervention blood glucose concentrations from 0 to 330 or 0 to 960 minutes post‐intervention compared to normal ad libitum conditions. Pre‐intervention glucose was quantified as the average glucose between −30 and −15 minutes. * and # represent a significant difference compared to females in P‐E and M‐D, respectively. ~, +, =, ¥ and ⱡ represent a significant difference compared to 6 hours with whole cage change (WCC), 6 hours with bedding retention cage change (BRCC), 6 hours with no cage change (NCC), ad libitum conditions and 16 hours with BRCC, respectively (P < 0.05, two‐way ANOVA with Holm‐Sidak post hoc tests). Data are mean ± SEM (n = 7 for males, n = 5‐7 for females in P‐E and n = 4‐7 for females in M‐D)

FIGURE 3

Effect of 6‐hour (6h) and 16‐hour (16h) fasts with different cage change methods on intraperitoneal glucose tolerance test (IPGTT) outcome in (A) males, (B) females in proestrous‐oestrous (P‐E) and (C) females in metoestrous‐dioestrous (M‐D). (D) Area under the curve (AUC)₁₂₀ for graphs A‐C. * and # represent a significant difference compared to females in P‐E and M‐D, respectively. ~, + and = represent a significant difference compared to 6 hours with whole cage change (WCC), 6 hours with bedding retention cage change (BRCC) and 6 hours with no cage change (NCC), respectively (P < 0.05, two‐way repeated‐measures ANOVA with Holm‐Sidak post hoc tests). Data are mean ± SEM (n = 7 for males, females in P‐E and females in M‐D)

FIGURE 4

Effect of oral gavage and oral glucose gels on 6‐hour (6h)‐fasted intraperitoneal glucose tolerance test (IPGTT) outcome following bedding retention cage changes (BRCC) in (A) males; (B) females in proestrous‐oestrous (P‐E); and (C) females in metoestrous‐dioestrous (M‐D). (D) Area under the curve (AUC)₁₂₀ for graphs (A‐C). * and # represent a significant difference compared to females in P‐E and M‐D, respectively. ~ represents a significant difference compared to oral glucose gels (P < 0.05, two‐way repeated‐measures ANOVA (A–C) and two‐way ANOVA) (D) with Holm‐Sidak post hoc tests). Data are mean ± SEM (n = 5 for males, females in P‐E and females in M‐D)

FIGURE 5

Effect of oral metformin and intraperitoneal (IP) exendin‐4 on 6‐hour (6h)‐fasted IP glucose tolerance test (GTT) outcome following bedding retention cage changes (BRCC). (A, B) IP exendin‐4 vs IP saline control in (A) males and (B) females. (D, E) Oral metformin versus sweetener gel control in (D) males and (E) females. (C and F) Area under the curve (AUC)₁₂₀ for graphs (A, B) and (D, E), respectively. * and # represent a significant difference compared to females and control, respectively (P < 0.05, two‐way ANOVA with Holm‐Sidak post hoc tests). Data are mean ± SEM (n = 4 for males and females)