Standard operating procedures for describing and performing metabolic tests of glucose homeostasis in mice

Abstract

The Mouse Metabolic Phenotyping Center (MMPC) Consortium was established to address the need to characterize the growing number of mouse models of metabolic diseases, particularly diabetes and obesity. A goal of the MMPC Consortium is to propose standard methods for assessing metabolic phenotypes in mice. In this article, we discuss issues pertaining to the design and performance of various tests of glucose metabolism. We also propose guidelines for the description of methods, presentation of data and interpretation of results. The recommendations presented in this article are based on the experience of the MMPC Consortium and other investigators.

Fig. 1.

Choosing a test of glucose metabolism in mice. For a given mouse model, the first step is to perform screening tests. The simplest test is to measure fasting insulin and glucose. In this example, mouse model “X” exhibits lower fasting glucose but normal insulin levels compared with control mice. The next screening test is to perform a GTT; in this example, mouse model “X” has improved glucose tolerance compared with control mice. ITTs can be used as an additional screening test if an effect on insulin action is suspected. If an improved glucose tolerance is thought to be due to enhanced pancreatic function, then a hyperglycemic clamp would be conducted. If the improvement is thought to be due to enhanced insulin action, then a hyperinsulinemic-euglycemic clamp would be conducted. Each of these tests can be followed up by more-specific analyses (isolated perifusion studies for islet function and/or stable isotope or metabolomic studies for glucose fluxes).

Fig. 2.

Comparison of whole-blood glucose meters and plasma glucose measurements. The regression line obtained when comparing glucose values obtained using the AlphaTRAK® (top panel) or the Accu-Chek Advantage (middle panel) whole-blood glucose monitors and plasma glucose values obtained using an enzymatic assay does not intersect the y-axis at zero. Comparison of glucose values obtained with each monitor (bottom panel) shows that the regression line also does not intersect the y-axis at zero.

Fig. 3.

Protocol for hyperinsulinemic-euglycemic clamps in mice. *Mice are fasted either overnight (14 to18 hours) or for shorter intervals (5 to 6 hours) prior to insulin infusion. A primed-continuous infusion of [3-³H]glucose is administered beginning either 120 or 90 minutes prior to the clamp period. Blood samples (in red) are obtained at the end of the equilibration period for assessment of basal parameters, such as basal glucose turnover and insulin concentration. The clamp period consists of a constant insulin infusion and a variable glucose infusion. Blood samples are obtained every 10 minutes for the measurement of glucose concentration. The variable glucose infusion is adjusted accordingly to maintain euglycemia. Saline-washed erythrocytes (RBCs), or an equiosmolar solution, are administered to prevent a fall in hematocrit. From t=80 to 120 minutes, blood samples are obtained for the measurement of steady-state clamp glucose turnover and other hormones and metabolites. A bolus of 2[¹⁴C]deoxyglucose can then be administered for the measurement of tissue-specific glucose uptake.

Fig. 4.

Methods for obtaining blood samples during hyperinsulinemic-euglycemic clamps in conscious mice. Blood can be obtained by cutting off the tip of the tail (left) or via a surgically implanted arterial catheter (right). Sampling from the cut tail requires restraint of both the mouse and the tail and handling of the tail throughout the experiment. When sampling from an arterial catheter, restraint is not necessary, and once the mouse is tethered to the swivel apparatus it is no longer handled.