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: Plasma Compartment Peripheral tissues glucose uptake Hepatic glucose production Exogenous glucose infusion (euglycemic) More glucose infusion = higher insulin sensitivity index Insulin - X +

The principles of a CLAMP:

Insulin resistance? Sensitive Resistant Severe resistance Insulin concentration Effect (%) 1 2

Differences in tissue-specific insulin sensitivity: EGP Insulin concentration Effect (%) BGU

Factual clamp data showing the main tissues: Periphery Liver Adipose tissue 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:

Double tracer CLAMP, tissue specific glucose uptake:

2-Deoxy glucose as glucose tracer:

Other analyses possible during CLAMP:

That is why choosing your infusion insulin concentration is very important: Conclusions: Insulin = 3 GIR control = 80 µmol/min GIR model X = 45 µmol/min 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!!!

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 : 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