2-NBDG as a fluorescent indicator for direct glucose uptake measurement

doi:10.1016/j.jbbm.2005.08.001

Journal of Biochemical and Biophysical Methods

Volume 64, Issue 3, 30 September 2005, Pages 207-215

https://doi.org/10.1016/j.jbbm.2005.08.001 Get rights and content

Abstract

Evaluation of glucose uptake ability in cells plays a fundamental role in diabetes mellitus research. In this study, we describe a sensitive and non-radioactive assay for direct and rapid measuring glucose uptake in single, living cells. The assay is based on direct incubation of mammalian cells with a fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) followed by flow cytometric detection of fluorescence produced by the cells. A series of experiments were conducted to define optimal conditions for this assay. By this technique, it was found that insulin lost its physiological effects on cells in vitro meanwhile some other anti-diabetic drugs facilitated the cell glucose uptake rates with mechanisms which likely to be different from those of insulin or those that were generally accepted of each drug. Our findings show that this technology has potential for applications in both medicine and research.

Introduction

Diabetes mellitus is one of the most prevalent and serious metabolic diseases and the principal cause of morbidity and mortality in the human. In diabetes, there is a failure to increase glucose uptake into peripheral tissues in response to insulin, leading to chronically elevated levels of glucose in the circulation [1]. A focus of current anti-diabetic medicine research is the development and screening of compounds with potential insulinomimetic effects to stimulate rate of cell glucose uptake [2]. Most studies on glucose uptake are commonly carried out using radiotracers such as 2-deoxy-d-[¹⁴C]glucose or 2-deoxy-d-[³H]glucose. However, there are several disadvantages associated with the radiotracer such as disposal of radioactive waste or radioactive cleanup. More importantly, it cannot directly measure glucose uptake in single, living cells [3]. We present an assay that is based on direct incubation of mammalian cells with a fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) followed by flow cytometric detection of fluorescence produced by the cells.

This is the first report, which combines a fluorescent glucose analog with flow cytometry technique that allowing direct and more sensitive measurement of glucose uptake cell by cell. In addition, there has been no method previously to directly acquire the relative fluorescent density of each cell, and we solved this problem by introducing a free software WinMDI to digitize flow cytometry plots for further statistical analysis. This technique is characterized by its simplicity and accuracy and can be extended to wide range of applications such as screening leading compounds for anti-diabetic drug candidates or offering a detection method to help provide insights into the mechanisms of glucose accumulation and metabolism in cells.

Section snippets

Cell culture

HepG2 human hepatocarcinoma cells and L6 rat skeletal muscle cells were cultured in MEM (Minimum Essential Medium) and α-MEM growth medium with 10% fetal bovine serum (FBS), respectively. Cells were maintained at 37 °C in a humidified 5% CO₂ environment.

Glucose uptake assay

Cells were plated at 1 × 10⁴/well in 96-well plates and used at subconfluence after 24 h preincubation. The authors strongly recommended that experiments be performed within 48 h. Longer incubation time, excessively confluent or too few cells will

Results and discussion

2-NBDG (Molecular Probes) is a new fluorescent derivative of glucose modified with a 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino group at the C-2 position (MW = 342.26) [4]. This product showed intense fluorescence at 542 nm when excited at 467 nm. Propidium Iodide (PI) is a nuclear dye known to permeate the cell membrane of dead cells while living cell is resistant to this dye [5]. Abnormal changes of PI fluorescence intensity, FSC (relative cell size or volume) or SSC (relative cell internal

Simplified description of the method and its applications

Evaluation of the ability of glucose uptake by organisms is one of the main aspects of diabetes research. We here describe a new system for the rapid and direct glucose uptake measurement with a fluorescent d-glucose analog 2-NBDG. Such non-radioactive tags are easier to visualize and do not pose a radiation hazard. Each single cell was considered as an individual sample in our experiments, we did not require destruction of the cells for each data point, and our experimental design was

Acknowledgments

The research was supported by the National Nature Science Foundation of China (grants 90209037). The authors wish to thank professors S. Zhao, Z.Y. Song and Z.W. Hu for the help with the manuscript.

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Measurement of glucose uptake and intracellular calcium concentration in single, living pancreatic β-cells
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Fat in all the wrong places
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A fluorescent compound for glucose uptake measurements in isolated rat cardiomyocytes
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There are more references available in the full text version of this article.

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Journal of Biochemical and Biophysical Methods

Abstract

Introduction

Section snippets

Cell culture

Glucose uptake assay

Results and discussion

Simplified description of the method and its applications

Acknowledgments

References (12)

Measurement of glucose uptake and intracellular calcium concentration in single, living pancreatic β-cells

J Biol Chem

A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli

Biochim Biophys Acta

A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin-V

J Immunol Method

Glucose uptake rates of single E. coli cells grown in glucose-limited chemostat cultures

J Microbiol Methods

Fat in all the wrong places

Nature

A fluorescent compound for glucose uptake measurements in isolated rat cardiomyocytes

Can J Physiol Pharmacol

Cited by (361)

Engineering vascularized skin-mimetic phantom for non-invasive Raman spectroscopy

Fluid shear stress enhances dendritic cell activation

Acrolein induces mitochondrial dysfunction and insulin resistance in muscle and adipose tissues in vitro and in vivo.

Hydrogen Sulfide Regulates Glucose Uptake in Skeletal Muscles via S-Sulfhydration of AMPK in Muscle Fiber Type-Dependent Way

Perfluorohexanesulfonic acid (PFHxS) induces oxidative stress and causes developmental toxicities in zebrafish embryos

The eIF4EBP-eIF4E axis regulates CD4<sup>+</sup> T cell differentiation through modulation of T cell activation and metabolism