Dr. Aileen King presents data showing that even small alterations to experimental protocol and choice of model can substantially impact both welfare and data interpretation when studying blood glucose homeostasis in mice. Using data from continuous glucose monitoring in mice, Dr. Aileen King discusses the myth that female mice present a more variable model due to the estrous cycle. Moreover, she demonstrates how different husbandry and protocol choices affect blood glucose concentrations in mice. Finally, Dr. King reflects on how this information can help design experiments using traditional methods of measuring blood glucose concentrations. Key Topics Include: - Understand how different male and female mice really are with regard to blood glucose homeostasis - Appreciate how small changes to protocols, that are often not described in methods, can affect outcome - Reflect on what we can and can’t change in our protocols and what we should consider when interpreting data To watch the webinar, go to: https://insidescientific.com/webinar/sex-drugs-and-protocol-how-researcher-choices-impact-experimental-outcomes-in-preclinical-diabetes-research/

1. Aileen King, PhD
Senior Lecturer in Pharmacology;
Diabetes Research Group
King’s College London
Sex, Drugs and Protocol:
How Researcher Choices Impact
Experimental Outcomes in
Preclinical Diabetes Research

2. Dr. Aileen King presents data showing how small alterations to
experimental protocol and choice of model can substantially
impact both welfare and data interpretation when studying
blood glucose homeostasis in mice.
Sex, Drugs and Protocol:
How Researcher Choices Impact
Experimental Outcomes in
Preclinical Diabetes Research

3. Sex, Drugs and Protocol:
How Researcher Choices
Impact Experimental
Outcomes in Preclinical
Diabetes Research
Aileen King
Diabetes Research Group
King’s College London
Copyright 2021 A. King and InsideScientific. All rights reserved.

4. Outline
Sex, Drugs and Protocol
A study using continuous blood glucose monitoring in mice
Part 1
Sex: How do female mice differ from males?
Part 2
Protocol: How variations in protocol affect experimental outcomes?
Part 3
Drugs: Does changing experimental protocol affect our ability to
detect drug effects?

5. Blood glucose
homeostasis
• Insulin promotes uptake and storage
of glucose into peripheral tissues
• Glucagon promotes glucose
production from the liver
• Blood glucose concentrations are
kept within the range of 4-7mM
• Diabetes develops due to an absolute
or relative deficiency of insulin
• The main symptom of diabetes is
hyperglycaemia

6. Animal models are an important
tool in diabetes research

7. Female mice are
underused in preclinical
diabetes research
• An assumption that estrous cycle
will lead to higher variability
• Estrogen has known effects on
beta cell biology
• Males tend to have a more
hyperglycaemic phenotype:
more suitable to test drugs?

8. Assessing blood glucose
homeostasis in mice
• Random blood glucose measurements
– Fixed, single time-point
measurements
– Requires handling and tail-prick
• Glucose tolerance tests
– Assesses glucose homeostasis under
stimulated conditions
– Mice are fasted and glucose bolus
administered
– Requires multiple handling and tail-
prick

9. HD-XG probe implanted
in aortic arch
Normoglycaemia measured
in unrestrained mice for
10-14 days
Overall blood glucose (mean, median,
total AUC, normal target range)
Glycaemic variability
Effect of common in vivo techniques
Continuous glucose monitoring increases
data resolution

10. Measurement of blood glucose concentrations
in unrestrained mice

11. Potential benefits of
continuous glucose
monitoring
• Welfare
– Conscious animals in normal state
– Minimised human presence and
stress reduction
– Reduced blood sampling
• Science
– Captures data that otherwise would be
missed e.g. excursions, night-time readings
– Can study impact of researcher intervention

12. Aims
• What are normal blood glucose
concentrations in mice and how
much do they fluctuate?
• Are blood glucose concentrations
in female mice more variable?
• What impact does varying
experimental protocol have on
outcomes?
• Are higher blood glucose
concentrations required to detect
drug effects?

13. What are normal blood glucose concentrations in mice?
Normal target range: 4.6-7.6mM
Median: 6.1mM
Normal target range: 5.2-8.7mM
Median: 6.9mM
Males Females

14. Sex: Male mice have higher blood glucose concentrations

15. Measuring glucose concentrations and variability
1. Mean absolute deviation:
how far each data-point
deviates from the median
2. CONGA-2: the standard
deviation of differences
between glucose values
every 2h
3. MAGE: average of
excursions above 1SD
24h median

16. CONGA-2 MAGE
MAD
Male mice have higher within-day variability regardless of
the estrous cycle or time of day
* vs females in P-E
# vs females in M-D

17. Male mice have higher within-day variability regardless of
the estrous cycle or time of day
* vs females in P-E
# vs females in M-D

18. Between-day variability:
Males have more variable blood glucose between days
MODD: Mean of daily differences

19. Between-animal variability: Individual male mice deviate
more from the cohort mean than individual female mice
Coefficient of variation for
all mice on individual days
vs the daily cohort average

20. Take-home message
Female mice have
lower blood glucose
concentrations than
males
Female mice have less
variable blood glucose
concentrations than
males
The estrus cycle does
not lead to significant
changes in blood
glucose concentrations

21. How do common
procedures
affect blood
glucose
concentrations?

22. Changing cage
increases blood
glucose
concentrations
for 1-2 hours
# vs baseline
* vs females

23. Increase in blood glucose concentrations in response to
simple in vivo procedures
# vs disturbance
Disturbance
Handling
Blood sampling
Intraperitoneal
injection

24. Glucose tolerance test (GTT) protocol
Fasting Handling
Glucometer
measurement
Glucose
administration
Ensure a stable blood glucose
and reduce variability
Weigh animals to
determine glucose dose
Baseline glucose value Determine glucose
tolerance
15-30 mins before GTT
6h or 16h before GTT Start of GTT
Immediately prior to GTT

25. Overall effect of
fasting, cage
changing, handling
and glucose injection
6h GTT with cage change and bedding retention
TIme of day
Blood
Glucose
(mM)
5
10
15
20
25
07:00 08:00 09:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00 18:00 19:00
Start of fast Pre-GTT
handling
Glucose injection
Refeeding

26. The increase in blood
glucose concentrations
seen during a GTT is
partially due to the
effects of the procedure
itself
Effect of
procedure
Effect of
glucose
bolus

27. Does altering
the GTT
protocol
affect welfare
and/or
outcome?
• Fasting
─How do you remove the food?
─How long to fast for?
• Glucose administration
─Route

28. No cage change
Food hopper emptied
Mouse remains in original cage
Bedding retention
Mouse transferred to new cage
Bedding from old cage is added
Whole cage change
Mouse transferred to new cage
New bedding
Ensures no food remnants in cage
Initiating the fast

29. Initiating the fast increases blood glucose concentrations
NCC No cage change
BRCC Bedding retention
cage change
WCC Whole cage change
BRCC Bedding retention
cage change
6h: fast started at 9am
16h: fast started at 6pm

30. How does the cage change affect the fasting outcome?
NCC No cage change
BRCC Bedding retention
cage change
WCC Whole cage change
BRCC Bedding retention
cage change
6h: fast started at 9am
16h: fast started at 6pm

31. Most effective fast: 16 hour
NCC No cage change
BRCC Bedding retention
cage change
WCC Whole cage change
BRCC Bedding retention
cage change
6h: fast started at 9am
16h: fast started at 6pm

32. Most effective fast: 16 hour
Least effective fast: no cage change
NCC No cage change
BRCC Bedding retention
cage change
WCC Whole cage change
BRCC Bedding retention
cage change
6h: fast started at 9am
16h: fast started at 6pm

33. Male mice fasted overnight become hypoglycaemic
NCC No cage change
BRCC Bedding retention
cage change
WCC Whole cage change
BRCC Bedding retention
cage change
6h: fast started at 9am
16h: fast started at 6pm

34. • Alterations to the fasting protocol can
affect:
─Efficiency of the fast
─Animal welfare
• Can the fasting protocol affect
outcome of the glucose tolerance test?
Fasting protocols
NCC No cage change
BRCC Bedding retention
cage change
WCC Whole cage change
BRCC Bedding retention
cage change
6h: fast started at 9am
16h: fast started at 6pm

35. Male mice fasted overnight have impaired glucose tolerance
NCC No cage change
BRCC Bedding retention
cage change
WCC Whole cage change
BRCC Bedding retention
cage change
6h: fast started at 9am
16h: fast started at 6pm

36. 6 hour fast (bedding retention) 16 hour fast (bedding retention)
Female mice fasted overnight show estrous-
related changes in GTT outcome

37. Whole cage change No cage change
Bedding retention
*
*
Whole cage change
Female mice subjected to a whole cage change show
estrous-related changes in GTT outcome

38. BRCC Bedding retention cage change
Take-home message
• Overnight fasting
─ Not recommended on welfare grounds
─ Impacts results in both male and female mice
• Whole cage change
─ Not recommended on welfare grounds
─ Could impact on results
• We recommend the 6h fast with bedding
retention cage change
─ Efficient fasting
─ Improved welfare

39. • Refinement of the fast
─6h rather than 16h fast
─Bedding retention in cage change
• What about the glucose administration?
Can we further refine the glucose tolerance test?

40. • Glucose mixed with
gelatin and flavouring
• Mouse trained to eat it
in less than 60 seconds
Mice can be trained to voluntarily ingest glucose gels
or gels containing drugs

41. • More physiologically accurate
─ Incretin system: oral glucose induces higher insulin secretion
• Less stress?
─ No restraint required however temporary separation required
Implications of mice voluntarily eating glucose gels

42. Males Females: proestrus-estrus Females: metestrus-diestrus
Voluntarily gel ingestions reduces glucose peak

43. Males
16 h overnight fast
i.p. glucose
Females
6 h fast
Voluntary glucose ingestion
Do we need the elevated peak in glucose to detect drug effects?

44. Metformin
Control
Metformin
Control
6h fast, bedding retention cage change, voluntarily ingestion of glucose gel
Effect of voluntary ingestion of metformin in a gel can be
observed in both males and females during a refined GTT
Females
Males

45. Take-home message
• Refined protocols can be used
to detect drug effects, despite
blunted glucose responses in
the control

46. • Female mice have lower blood glucose
concentrations but also lower variability
• Estrus does not affect random blood
glucose concentrations
• Estrus does not affect glucose tolerance
when using the most refined protocols
• Fasting mice overnight is not only a welfare
concern but can also can impact results
• Elevated glucose responses are not
required to detect drug effects
Summary

47. • Female mice should be included in
studies of blood glucose homeostasis
• Subtle alterations to protocols can
affect results
• Be consistent and report protocols
accurately!
Conclusion

48. Acknowledgements
• Matilda Kennard
• Manasi Nandi
• Data Sciences International
• British Pharmacology Society

49. • Want to learn more about Aileen King’s
research?
Visit: www.kcl.ac.uk/people/aileen-king
• Want to learn more about DSI’s
implantable telemetry techology?
Visit: www.datasci.com
Thanks for participating!
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