Disease Model Core Metabolic Phenotyping: which parameters
do we (or need to) measure in Glucose Handling?
Peter J Voshol, PhD
Director of Disease Model Core
Senior Research Associate: Integrative Physiology
Disease Model Core Metabolic Phenotyping: which parameters
do we (or need to) measure in Glucose Handling?
Discuss the following:
1) Glucose-phenotyping: Using CD36-/- as example
2) Discussing specific considerations and methods
3) Suggestion for SOP guides for phenotyping
CD36…???
Fatty acid translocase, also known as CD36:
1) A receptor for several ligands, including oxidized LDL
and long chain fatty acids.
2) CD36 is abundant in peripheral tissues active in fatty
acid metabolism, such as adipose tissue and skeletal
and cardiac muscle, where it is involved in high-affinity
uptake of fatty acids.
CD36, fatty acid translocator:
Knockout leads to altered disposition of FFA’s
What about Glucose Metabolism?
Higher insulin sensitivity during a ITT (fixed dose) on chow fed
mice.
What about a GTT?
The dose is important:
Am J Physiol Endocrinol Metab 297: E849–E855, 2009.
What about our HFD model in real life?
Same dose of glucose with ~10g BW difference:
The effect is in Insulin!
The AUC calculated and corrected for baseline values.
Is there a difference IP of ORAL GTT?
Effect of fasting on baseline glucose and insulin levels:
Effect of fasting on GTT: glucose and insulin levels:
Am J Physiol Endocrinol Metab 295:E1323-E1332, 2008.
Same dose of glucose with ~10g BW difference:
The effect is in Insulin!
Similar to Human CLAMP:
The principles of a CLAMP:
More glucose infusion
=
higher insulin
sensitivity index
Exogenous glucose
infusion
Insulin
-
X
Insulin
+
Plasma
Compartment
Hepatic glucose (euglycemic)
production
Peripheral tissues
glucose uptake
The principles of a CLAMP:
Insulin resistance?
120
Effect (%)
100
2
80
1
60
40
Sensitive
Resistant
20
Severe resistance
0
0
5
10
Insulin concentration
15
Differences in tissue-specific insulin sensitivity:
120
Effect (%)
100
80
60
40
EGP
20
BGU
0
0
2
4
6
8
Insulin concentration
10
12
Factual clamp data showing the main tissues:
Periphery
Adipose tissue
Liver
Am J Physiol Endocrinol Metab 291: E1360–E1364, 2006.
The basic CLAMP (no tracer kinetics)
The tracer kinetic CLAMP
Principle of tracer dilution
Remember the kinetics:
The calculations:
Insulin-stimulated tracer dilution
Insulin-stimulated kinetics:
Changes in blood tracer dilutions:
A word on Euglycemia:
Normal concentration of glucose in the blood. Also
called normoglycemia.
The ‘normal’ or baseline fasted glucose concentration
measured in your mouse model!
Euglycemic
That is why choosing your infusion insulin concentration is very
important:
That is why choosing your infusion insulin concentration is very
important:
That is why choosing your infusion insulin concentration is very
important:
Double tracer CLAMP, tissue specific glucose uptake:
2-Deoxy glucose as glucose tracer:
Other analyses possible during CLAMP:
Conclusions:
Insulin = 3
GIR control = 80 µmol/min
GIR model X = 45 µmol/min
Insulin-mediated effect (%)
That is why choosing your infusion insulin concentration is very
important:
120
Control
100
Animal X
80
60
40
20
0
0
2
4
6
8
-20
Insulin concentration
Model X is insulin resistant compared to controls!!!
Insulin = 6
GIR control = 100 µmol/min
Gir model x = 100 µmol/min
Model X has equal insulin sensitivity compared to control!!!
10
12
Example:
Diabetes 55:390–397, 2006
Example
Diabetes 55:390–397, 2006
And fasting has an effect of course:
Mice get peripheral insulin sensitivity upon fasting!
Background breeding effect:
Diabetes 57:1790–1799, 2008
Back to CD36-/- mouse model:
Improved peripheral glucose metabolism
J.Lipid Res. 2003. 44: 2270–2277.
CD36-/-:
But decreased hepatic insulin sensitivity.
CD36-/-:
Due to TG accumulation in CD36-/- mice
Conclusion on glucose metabolism in CD36-/- mice:
What SOP can be suggested to run:
1)Lipid metabolism
2)Glucose metabolism
3)Energy balance
Basal lipid-phenotyping
HFD-induced lipid-phenotyping
Basal glucose-phenotyping
HFD-induced glucose-phenotyping
Other considerations:
1)Temperature, housing, experiments?
2)Aging effects, normal diet and diet interventions?
3)Background strain differences, cross breeding
4)Littermates (in relation to 1, 2 and 3)
Thank you