Assessment of Tissue-Specific Glucose Uptake: Teaching an Old 2-DOG New Tricks

A major function of insulin in skeletal muscle, heart, and white and brown adipose tissue is to stimulate cellular glucose uptake (1). Tissue-specific measurements of insulin-stimulated glucose uptake are useful for dissecting the mechanisms underlying abnormalities detected by tests of whole-body glucose homeostasis, such as the glucose or insulin tolerance test. Several methods using isotope-labeled, nonmetabolizable glucose tracers are available for assessing tissue-specific glucose uptake in vivo, including [¹⁸F]fluorodeoxyglucose (FDG) positron emission tomography (2) and bolus injection of [¹⁴C]2-deoxyglucose (2-DOG) during a hyperinsulinemic-euglycemic clamp (3). These tracers behave like glucose and enter the cell by facilitated diffusion via GLUT transporters. Following uptake, 2-DOG is phosphorylated by hexokinase but cannot be metabolized further by the glycolytic machinery. The phosphorylated glucose tracer is therefore trapped in cell types that do not express glucose-6-phosphatase, enabling measurement of tissue-specific glucose uptake. However, these techniques require specialized equipment, demand procedural expertise, and are generally low throughput (4). They also measure glucose uptake under one condition (e.g., basal or insulin stimulated), limiting data interpretation.

In this issue of Diabetes, Cutler et al. (5) establish a new method to quantify basal and insulin-stimulated glucose uptake, termed the “Dual Tracer Test.” In the Dual Tracer Test, sequential injections of [¹⁴C]2-DOG and insulin plus [³H]2-DOG are administered to the same anesthetized mouse. The authors evaluated the Dual Tracer Test in various tissues from insulin-sensitive and insulin-resistant C57BL/6J mice, finding that for some tissues, but not all, correcting insulin-stimulated glucose uptake to basal uptake reduced the variability between mice compared with analysis of the insulin-stimulated values alone. Best practices for performance and interpretation of the Dual Tracer Test are provided, and its limitations are discussed. Finally, a novel mouse model with reduced systemic GLUT4 expression was used for further validation, leading to the potentially novel observation of insulin resistance without compensatory hyperinsulinemia, which will require further exploration.

To perform the Dual Tracer Test, mice are anesthetized with pentobarbital and tail-bleed access is established. An initial retroorbital injection of [¹⁴C]2-DOG is followed by blood sampling at 2, 15, and 30 min, and at 40 min, a second bolus of [³H]2-DOG and insulin (0.75 units/kg fat-free mass, a pharmacologic/maximal dose) is administered, and blood is sampled again at 2, 15, 30, and 40 min. Tissues are collected and standard analytical procedures used to determine plasma tracer kinetics and tissue 2-DOG–6-phosphate content. Tissue uptake of [¹⁴C]2-DOG provides a readout of basal glucose uptake, while tissue uptake of [³H]2-DOG represents insulin-stimulated glucose uptake.

The authors performed the Dual Tracer Test in chow-fed and high-fat diet (HFD)–fed mice, which revealed HFD-induced insulin resistance to glucose uptake in soleus and white adipose tissue and a trend in brown adipose tissue but no differences in red quadriceps, extensor digitorum longus, diaphragm, or heart. HFD-fed mice displayed decreased glucose uptake in soleus and subcutaneous adipose tissue in both basal and insulin-stimulated states, highlighting the value of measuring glucose uptake in both conditions. Indeed, correcting insulin-stimulated glucose uptake to basal measurements from the same mouse reduced the coefficient of variation between mice compared with analysis of insulin-stimulated uptake alone, particularly for adipose tissues. Thus, the authors propose that the Dual Tracer Test is useful for reducing the group sizes required to detect subtle phenotypes, such as in studies using outbred mice to identify regions of the genome associated with differences in insulin-stimulated glucose uptake.

In addition to validating their new approach in a dietary model of insulin resistance, the authors applied the Dual Tracer Test to a unique knock-in mouse strain with an ∼75% reduction in GLUT4 protein content. Using this model, the authors demonstrated reduced insulin-stimulated glucose uptake in skeletal muscle, white and brown adipose tissue, and diaphragm and brain. Thus, studies in a model of genetically perturbed glucose transport validated the utility of the Dual Tracer Test. Interestingly, these mice did not display fasting hyperinsulinemia, which may relate to preserved hepatic insulin sensitivity and normal fasting glucose concentration. This potential novel observation of insulin resistance without hyperinsulinemia is exciting and will require more detailed characterization than was possible here to establish its importance.

The Dual Tracer Test has numerous limitations, which are readily acknowledged by the authors but are critical to highlight for investigators considering its use. There are several physiological considerations that will impact data interpretation. The use of pentobarbital anesthesia causes hyperglycemia (5), hyperinsulinemia (6), and insulin resistance in liver and skeletal muscle (7,8). The basal [¹⁴C]2-DOG tracer is also not fully cleared from plasma by the time of insulin injection. These effects of anesthesia and incomplete basal tracer clearance will both lead to overestimation of basal tissue glucose uptake and underestimation of tissue insulin sensitivity, although the authors show that the effect of incomplete basal tracer clearance is only relevant for very insulin-sensitive tissues, such as heart, brown adipose tissue, and possibly soleus. Moreover, the maximal insulin dose will cause hypoglycemia in insulin-sensitive animals, decreasing tissue glucose uptake during the insulin-stimulated period (9). This effect will cause underestimation of insulin sensitivity in very insulin-sensitive animals and may make it more challenging to identify subtle differences in insulin sensitivity. Precise timing for collection of the 2-min blood samples is critical for accurate determination of plasma tracer kinetics; any delay will introduce significant error to the extrapolated initial concentration of tracer in blood that is used to calculate tissue-specific glucose uptake. There are also logistical considerations. Performing multiple retroorbital injections may not be permitted by some institutional animal care and use committees (10). Although the Dual Tracer Test requires less specialized equipment and procedural expertise than the hyperinsulinemic-euglycemic clamp, its throughput may still be limited by costs associated with radioisotope acquisition, use, and disposal in animals as well as the analytical effort and time required for sample processing and liquid scintillation counting. Carefully executed glucose and insulin tolerance tests should not be ignored when considering simple approaches to ascertain differences in insulin resistance.

Despite these limitations, the Dual Tracer Test fulfills an unmet need in the mouse metabolic phenotyping toolkit. Other physiological functions of insulin, such as suppression of hepatic glucose production and adipose tissue lipolysis, are frequently assessed in both basal and hyperinsulinemic conditions, and the Dual Tracer Test fills a gap by facilitating analogous assessment of basal and insulin-stimulated tissue glucose uptake. Although the classic hyperinsulinemic-euglycemic clamp combined with [³H]glucose infusion provides a measure of basal and insulin-stimulated whole-body glucose uptake in the same mouse, it does not provide the tissue-specific information that the Dual Tracer Test provides. In theory, the Dual Tracer Test could be combined with the classic clamp, but this would preclude simultaneous use of [³H]glucose to trace whole-body glucose turnover. Ultimately, all available tools for assessing insulin action in mice possess limitations that require thoughtful experimental design. To that end, the authors describe and propose specific data that must be collected and reported for proper interpretation of Dual Tracer Test results by the research community. Investigators, journal editors, and reviewers should pay keen attention to that section.