Cellular binding and uptake of fluorescent glucose analogs 2-NBDG and 6-NBDG occurs independent of membrane glucose transporters

Highlights

• Glucose uptake rates are a key metabolic parameter for cell growth.

• Fluorescent glucose analogs are commonly used to monitor glucose uptake.

• Murine L929 fibroblasts import glucose exclusively via the Glut1 transporter.

• Multiple methods of Glut1 inhibition fail to block fluorescent analog uptake.

• Fluorescent glucose analogs enter cells by transporter-independent mechanisms.

Abstract

The classical methods for determining glucose uptake rates in living cells involve the use of isotopically labeled 2-deoxy-d-glucose or 3-O-methyl-d-glucose, which enter cells via well-characterized membrane transporters of the SLC2A and SLC5A families, respectively. These classical methods, however, are increasingly being displaced by high-throughput assays that utilize fluorescent analogs of glucose. Among the most commonly used of these analogs are 2-NBDG and 6-NBDG, which contain a bulky 7-nitro-2,1,3-benzoxadiazol-4-yl-amino moiety in place of a hydroxy group on d-glucose. This fluorescent group significantly alters both the size and shape of these molecules compared to glucose, calling into question whether they actually enter cells by the same transport mechanisms. In this study, we took advantage of the well-defined glucose uptake mechanism of L929 murine fibroblasts, which rely exclusively on the Glut1/Slc2a1 membrane transporter. We demonstrate that neither pharmacologic inhibition of Glut1 nor genetic manipulation of its expression has a significant impact on the binding or uptake of 2-NBDG or 6-NBDG by L929 cells, though both approaches significantly impact [³H]-2-deoxyglucose uptake rates. Together these data indicate that 2-NBDG and 6-NBDG can bind and enter mammalian cells by transporter-independent mechanisms, which calls into question their utility as an accurate proxy for glucose transport.

Introduction

Glucose is a common catabolic fuel for the majority of mammalian cell types. Because the rate of glucose uptake is a key indicator of metabolic status, it is frequently assessed in studies across fields of biomedical science, including those focused on normal physiology and disease conditions such as cancer [1]. In vivo evaluation of glucose uptake is dominated by positron emission tomography using the tracer 2-[¹⁸F]-fluoro-2-deoxy-d-glucose (FDG-PET), which has become a mainstay for detection of tumor metastasis in medical oncology [2]. Biochemical methods for routine laboratory measurements of glucose uptake in living cells similarly rely on isotopic labeling and are considered the gold standard for this assay.

The most common in vitro assays for glucose uptake utilize radiolabeled forms of either 3-O-methyl-d-glucose (3-OMG) or 2-deoxy-d-glucose (2-DG) [3]. 3-OMG is not further metabolized and has the advantage of measuring only the transport process; however, the rapid exchange of glucose requires very short incubation times to accurately measure transporter activity, limiting its application in some studies. It has, therefore, become more common to utilized 2-DG to measure glucose uptake. This analog is transported and undergoes the first metabolic step of phosphorylation, which traps the radiolabeled 2-DG-6-phosphate in most cells. In nearly all cases, the transport step is significantly slower than the phosphorylation step, which allows radiolabeled 2-DG accumulation to serve as an accurate measure of transporter activity [4].

Despite their time-tested utility, the classical methods cited above are increasingly being displaced by fluorescence-based assays that utilize analogs of glucose in which one of the hydroxyl groups is replaced with a fluorophore that is itself similar in size or larger than glucose [5]. Among the various fluorescent analogs of glucose that are commonly used for these assays, 2-NBDG has emerged as the most popular in the literature [[6], [7], [8]]. This molecule has a bulky 7-nitrobenzofurazan fluorophore attached to d-glucosamine in place of the endogenous 2-hydroxy group. Though early work with 2-NBDG clearly demonstrate that it is taken up by bacteria and mammalian cells alike, very little effort has been expended to demonstrate that it enters cells by a mechanism that accurately mimics actual glucose uptake. Studies that have shown some impact of d-glucose on the rate of 2-NBDG uptake in a single cell line have been deemed sufficient to demonstrate its broader viability as a measurement of glucose uptake across all mammalian cell lines [9]. However, given the known mechanism of hexose transport across the plasma membrane, which involves both electrostatic and steric selectivity, it seems intuitively unlikely that 2-NBDG or any other fluorescent analog is actually transported in similar fashion [10,11].

One of the challenges in determining the mode by which 2-NBDG or other fluorescent analogs are taken up by mammalian cells is the complexity of glucose uptake mechanisms available to many cell types. Three distinct classes of eukaryotic sugar transporters have been characterized: (1) the recently discovered SWEET family, primarily responsible for intra- and intercellular transport; (2) the well-characterized passive glucose transporters of the GLUT/SLC2A family; and (3) the active sodium-glucose linked symporters of the SGLT/SLC5A family [12,13]. Fourteen different members of the SLC2A family and six members of the SLC5A family exist within the human genome, many of which are broadly distributed in expression across different tissues [14,15]. The overlapping expression of these transporters in any given cell type make the determination of which one is predominant in glucose uptake a challenging matter to dissect experimentally. The development of selective inhibitors that are able to distinguish among different glucose transporters has provided some help in this respect, as has the advent of genetic technologies capable of selective silencing the expression of single genes [16,17]. For the most part, however, the specificity regarding which transport system is being used for glucose absorption by a given cell type is rarely assessed in any significant detail when glucose uptake is being evaluated.

Of the various SLC2A family members that have been characterized in the literature, SLC2A1/GLUT1 stands out as perhaps the best understood due to its broad expression across different cell types and well-defined transport kinetics [11,18]. Furthermore, the protein structure for this transporter has been defined by X-ray crystallography, yielding additional insights into the mechanism by which it transports glucose across the plasma membrane [19]. The prominent role that GLUT1 plays in the hypoxic response and cancer metabolism has spurred further interest in its expression and regulation, as well as the generation of selective inhibitors that can block its transport function without significantly affecting other physiologically important glucose transporters [16,20,21]. In addition to their therapeutic potential, these drugs present helpful tools for dissecting the role of GLUT1 in basic research applications.

In this study, we systematically evaluated the ability of the mouse Slc2a1/Glut1 to bind and transport 2-NBDG and its structural isomer, 6-NBDG, in L929 fibroblasts. Compared to many other mammalian cell types, the L929 mouse fibroblast line is relatively simple in its mode of glucose uptake due to the fact that expresses only the Glut1 facilitative glucose transporter [22]. We leveraged this simple system to evaluate the impact of pharmacologic inhibition and genetic manipulation of Glut1 on 2-NBDG and 6-NBDG transport kinetics. Our results suggest that L929 cells bind to both glucose analogs and actively accumulate 2-NBDG, as expected, but do so by undefined mechanisms that largely fail to involve Glut1 or any other glucose transporter. These findings suggest that the uptake of fluorescent glucose analogs should be interpreted with great caution since they may not represent an accurate proxy for glucose transport in mammalian cells.