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Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

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Published on: April 6, 2022

Single Cell Glucose Uptake Assays: A Cautionary Tale.

Linda V Sinclair¹, Celine Barthelemy², Doreen A Cantrell¹

¹Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, Dundee, DD1 4HN, UK.

Immunometabolism

|September 4, 2020

Summary

The fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) is not a reliable measure of glucose transport in T cells. Its uptake does not correlate with actual glucose transport capacity in these immune cells.

Keywords:

2-NBDG T cells T lymphocytes glucose uptake

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A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

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A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Published on: August 12, 2016

Area of Science:

Immunology
Cellular Metabolism
Biochemistry

Background:

Assays for single-cell immune cell metabolic state and nutrient uptake are crucial.
2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) is a fluorescent glucose analog used to study glucose uptake.
2-NBDG is validated for Glut2 but its selectivity for T cell glucose transporters (Glut1, Glut3) and non-transporter binding is unexplored.

Purpose of the Study:

To investigate the specificity of 2-NBDG labeling as a readout for glucose transport in T lymphocytes.
To assess whether 2-NBDG uptake accurately reflects glucose transport capacity in T cells.
To determine if 2-NBDG binds to T cells independently of glucose transporters.

Main Methods:

Comparison of flow cytometric 2-NBDG staining with radiolabeled glucose transport assays in murine T cells.
Assessment of 2-NBDG uptake inhibition by competitive substrates and glucose transporter inhibitors.
Evaluation of 2-NBDG's ability to competitively inhibit glucose uptake.

Main Results:

Significant discordance observed between glucose transport capacity and 2-NBDG labeling in T cells.
2-NBDG uptake in murine T cells was not inhibited by known glucose transport inhibitors or competitive substrates.
2-NBDG did not competitively block glucose uptake in T cells.

Conclusions:

2-NBDG uptake alone is not a reliable tool for assessing cellular glucose transport capacity in T lymphocytes.
The mechanism of 2-NBDG uptake in T cells may not involve canonical glucose transporters.
Further research is needed to validate alternative methods for measuring T cell glucose uptake.