11/23/2019 Comparison of Glucose Uptake Assay Methods

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Comparison of Glucose Uptake Assay

Methods

Promega Corporation

Abstract
This article describes the various methods available to measure glucose uptake and lists the
advantages and disadvantages of each assay. Among these assay is the new non-radioactive
Glucose Uptake-Glo™ Assay, a simple and sensitive luminescent-based method for use with
multiwell plates.

Introduction
Glucose is the primary source of energy for many organisms, and the uptake of glucose is a
critical process. Glucose is transported across the cell’s membrane and trapped by being
phosphorylated. In mammalian cells, this is performed by a family of glucose transporters
(GLUT) and a few intracellular hexose kinases. Note that measuring glucose uptake is not the
same as measuring glucose consumption. Glucose uptake occurs on a rapid time scale of 10
minutes or less and specifically measures transporter activity, whereas changes in glucose
concentration involve a multitude of pathways and typically take several hours.

Why Measure Glucose Uptake?

We can learn several things from measuring glucose uptake. Changes in glucose uptake can
reflect overall changes in metabolism, but there are many specific processes as well. In cancer
cells, measuring glucose uptake can monitor the overexpression of glucose transporters or
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identify glucose transporter inhibitors. With fat and muscle cells, changes in GLUT4
translocation upon insulin stimulation can be observed by measuring glucose uptake. In
immunologically relevant cells, measuring glucose uptake can be used to follow the
transformation of certain cell types from one stage to another.

System Model Cells Purpose

3T3-L1, 3T3-
Fat and muscle
L1-MBX, L6, Monitor GLUT4 translocation in response to insulin
cells
C2C12

Immunologically T cells and

Monitor cell activation
relevant cells macrophages

Any cancer Monitor glucose transporter overexpression, identify

Cancer cells cell (e.g., glucose transporter inhibitors or measure general
HCT116) changes in metabolism

Traditional Glucose Uptake Methods

Available glucose uptake assays differ by the substrate for transport. Since glucose itself is
shuttled into a variety of pathways after transport, non-metabolizable glucose analogs must be
used instead. One type is the fluorescently tagged 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-
4-yl)amino)-2-deoxyglucose). This probe accumulates in the cell and works well for imaging,
but because it is much larger than glucose, there is concern that it is not transported in the
same way and hence may not be an accurate indicator of glucose transporter activity. Another
glucose analog is 2DG (2-deoxyglucose). This molecule gets transported and phosphorylated
just like glucose, but since it is not acted upon significantly by other cellular enzymes, it
accumulates in the cell as 2DG6P (2-deoxyglucose-6-phosphate).

Assay Principle Advantages Disadvantages

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Assay Principle Advantages Disadvantages

Requires multiple
wash steps
Intracellular accumulation Requires handling
Radioactivity Sensitive
of 3H-2DG6P and disposal of
radioactive
materials

Requires multiple
sample
Enzymatic detection of
Fluorescence Non-radioactive preparation steps
2DG6P
Narrow window of
detection

Requires multiple
Non-radioactive sample
Enzymatic detection of
Absorbance Can detect very preparation steps
2DG6P
low 2DG6P Narrow window of
detection

Doesn't work well

Intracellular accumulation in plate formats
Works well for
2-NBDG of a fluorescently labelled May not correctly
imaging
glucose analog reflect transporter
activity

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Assay Principle Advantages Disadvantages

Sensitive
Non-radioactive
Can detect very
low 2DG6P with
large assay
Enzymatic detection of Not applicable for
Luminescence window
2DG6P cell imaging
Direct in-well
detection with
no wash steps
Scalable for high
throughput

For a long time, the gold standard for measuring glucose uptake has been the radio-labeled
analog 3H-2DG and the measurement of accumulated 3H-2DG6P (1). Although the method is
sensitive and works well, it requires multiple wash steps. Many researchers do not use this
method because they want to avoid the handling and disposal of radioactive material.
Commercial non-radioactive methods use non-radiolabeled 2DG and measure the
accumulation of 2DG6P through the action of G6PDH (glucose-6-phosphate dehydrogenase)
(2). This enzyme oxidizes 2DG6P to form NADPH, which can then be used to generate probes
for absorbance or fluorescence assays. Both assays are amenable to multiwell plate formats,
but they require multiple processing steps. Due to a cycling detection reaction, the absorbance
assay is most sensitive and can detect very low amounts of 2DG6P. However, both assays have
small signal windows (i.e., signal above no 2DG6P background) that limit their use.

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Figure 1. Comparison of enzymatic detection assays. A titration of 2DG6P was assayed with the
luminescent, uorescent and absorbance methods. The results are reported as the signal divided by the
no-2DG6P control.

Figure 2. Comparison of radioactive and luminescent assays. Glucose uptake by 20,000 or 50,000

HCT116 cells initiated with 1mM 2DG for 10 minutes was measure with luminescent (Panel A) and
radioactive (Panel B) assays. Cytochalasin B (50µM) was added to determine assay background.
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Glucose Uptake-Glo™ Assay

We have developed a luminescent glucose uptake assay that is based on the same principle as
the aforementioned assays: The accumulation of 2DG6P and detection through the enzymatic
action of G6PDH (3). The luminescent assay has a number of advantages:

Similar sensitivity to the radioactive assay but without any wash steps or handling and
disposal of radioactive material.
Larger signal window than the absorbance and fluorescence assays, making it easier to
detect small changes and requiring less assay optimization.
Amenable to high-throughput screening due to fewer false hits and steady glow-type
signals.

The luminescent assay can detect glucose uptake from as few as 5,000 cells with a greater
than threefold signal-to-background ratio, and the signal remains linear up to at least 50,000
cells. The luminescent assay is the best combination of simplicity and sensitivity available for
a non-radioactive multiwell plate method.

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Figure 3. Protocol comparison of luminescent, uorescent and radioactive assays. Equivalent

preprocessing steps are colored gray.

References
1. Yamamoto, N. et al. (2015) Measurement of glucose uptake in cultured cells. Curr. Protoc.
Pharmacol. 71, 12.14.1–26.
2. Saito, K. et al. (2011) An enzymatic photometric assay for 2-deoxyglucose uptake in
insulin-responsive tissues and 3T3-L1 adipocytes. Anal. Biochem. 412, 9–17.

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3. Valley, M.P. et al.(2016) A bioluminescent assay for measuring glucose uptake. Anal.

Biochem. 505, 43–50.

Glucose Uptake-Glo™ Assay

A simple, non-radioactive assay to measure glucose uptake inside cells.

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Abstract

Introduction

Why Measure Glucose Uptake?

Traditional Glucose Uptake Methods

Glucose Uptake-Glo™ Assay

References

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