Acclimation Prior to an Intraperitoneal Insulin Tolerance Test to Mitigate Stress-Induced Hyperglycemia in Conscious Mice

Summary

Stress-induced elevations in glucose levels can confound interpretation of data derived from a conscious intraperitoneal insulin tolerance test in mice. In this article, we describe a method to acclimate the mice to handling, injections and blood sampling prior to performing the insulin tolerance test in order to limit stress-induced hyperglycemia.

Abstract

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that involves measurement of blood glucose levels over time following a single intraperitoneal injection of insulin. Given that it is performed in the conscious state and blood is often collected via a tail snip, it has the potential to elicit a stress response from animals due to anxiety associated with handling and blood collection. As such, a stress-induced rise in blood glucose can occur, making it difficult to detect and interpret the primary endpoint measure, namely an insulin-mediated reduction in blood glucose. This has been seen in many mouse strains, and is quite common in diabetic db/db mice, where glucose levels can increase, rather than decrease, after insulin administration. Here, we describe a method of acclimating mice to handling, injections and blood sampling prior to performing the insulin tolerance test. We find that this lowers stress-induced hyperglycemia and results in data that more accurately reflects whole body insulin sensitivity.

Introduction

Metabolic tests in rodents are routinely performed to assess various parameters that regulate glucose homeostasis¹. The gold standard for assessing whole-body insulin action in vivo is the hyperinsulinemic-euglycemic clamp². This test involves administration of insulin to raise circulating insulin levels while glucose is infused to maintain euglycemia. The glucose infusion rate required to maintain euglycemia is indicative of insulin action. While it is a powerful tool in metabolic research, the clamp technique in mice is technically challenging and labor intensive, and thus is not well suited as an initial screening too....

Protocol

All methods described here have been approved by the VA Puget Sound Health Care System’s Institutional Animal Care and Use Committee.

NOTE: Local requirements for monitoring and/or intervention of animals that experience hypoglycemia may differ from those described here.

1. Fasting (t= -210 min)

1. After the dark cycle has ended, transfer mice to a new cage with non-nutritious bedding such as cellulose or paper bedding (not corn-cob bedding, which will affect metabolic endpoints if consumed by mice¹³). Provide mice with ad libitum access to water throughout the fasting perio....

Results

Figure 2A and Figure 3A are representative data (individual and mean data, respectively) showing a paradoxical rise in blood glucose levels in diabetic db/db mice in the 15 minutes following insulin administration, consistent with stress-induced hyperglycemia. Note, no rise in blood glucose was evident in the control non-diabetic littermates (db/+ or +/+) that underwent the same procedure. To determine whether acclimation to the ITT procedure was effective in mi.......

Discussion

The hyperinsulinemic-euglycemic clamp is considered the gold standard for assessing insulin action in vivo. Modifications to the methodology for performing the clamp have resulted in the technique being done in conscious, unrestrained mice² that have been previously catheterized using a two-catheter system¹⁴ to enable blood sampling via the carotid artery and infusions via the jugular vein. This limits the need to handle or restrain mice during the procedure, thereby reduci.......

Materials

Name	Company	Catalog Number	Comments
Dextrose-50	Pfizer Injectables	00409-6648-16	For use if mouse experiences hypoglycemia.
Gauze pads	Fisher Scientific	22037907	To dislodge blood clot on the tail tip.
Glucometer	Accu-Chek	M001_us	To measure blood glucose.
Gram scale			To measure body weight.
Insulin (Novolin R)	Novo Nordisk	0169-1833-11	For injection.
Insulin syringes	VWR	BD-329461	For injections.
Minute timer
Sterile 20 G needle	VWR	BD-305175	For tail snip.
Sterile saline	Lifeshield	1261699	For injections.
Surgical scissors	Fine Science Tools	14088-10	For tail snip.
Test strips	Accu-Chek	06908217001_us	To measure blood glucose.