Genetic evidence that uptake of the fluorescent analog 2NBDG occurs independently of known glucose transporters

Lucas J D’Souza; Stephen H Wright; Deepta Bhattacharya

doi:10.1371/journal.pone.0261801

. 2022 Aug 24;17(8):e0261801. doi: 10.1371/journal.pone.0261801

Genetic evidence that uptake of the fluorescent analog 2NBDG occurs independently of known glucose transporters

Lucas J D’Souza ¹, Stephen H Wright ², Deepta Bhattacharya ^1,^*

Editor: Hodaka Fujii³

PMCID: PMC9401136 PMID: 36001583

Abstract

The fluorescent derivative of glucose, 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]-D-glucose (2NBDG), is a widely used surrogate reagent to visualize glucose uptake in live cells at single cell resolution. Using CRISPR-Cas9 gene editing in 5TGM1 myeloma cells, we demonstrate that ablation of the glucose transporter gene Slc2a1 abrogates radioactive glucose uptake but has no effect on the magnitude or kinetics of 2NBDG import. Extracellular 2NBDG, but not NBD-fructose was transported by primary plasma cells into the cytoplasm suggesting a specific mechanism that is unlinked from glucose import and that of chemically similar compounds. Neither excess glucose nor pharmacological inhibition of GLUT1 impacted 2NBDG uptake in myeloma cells or primary splenocytes. Genetic ablation of other expressed hexose transporters individually or in combination with one another also had no impact on 2NBDG uptake. Ablation of the genes in the Slc29 and Slc35 families of nucleoside and nucleoside sugar transporters also failed to impact 2NBDG import. Thus, cellular uptake of 2NBDG is not necessarily a faithful indicator of glucose transport and is promoted by an unknown mechanism.

Introduction

Glucose is a critical nutrient for fueling cellular metabolism. After its import, glucose can be catabolized through a multitude of metabolic pathways. For example, glucose can be broken down by glycolysis into pyruvate that in turn is oxidized to fuel the tricarboxylic acid (TCA) cycle and generate ATP [1]. Glucose is also important for generating intermediate sugars which serve as precursors for the synthesis of nucleotides, fatty acids, and glycosylation sugars [2]. Glucose uptake and catabolism are key defining features of cells, distinguishing between developmental stages of lineages, tumors versus normal tissues, and activation status.

Definitive methods to measure glucose import have classically involved the use of isotope-labelled derivatives of glucose [3–5]. A shortcoming to these compounds, however, is their rapid breakdown or export from the cell, and an inability to resolve glucose uptake at single cell resolution. Fluorescent derivatives of glucose, which allow for visualization and flow cytometric estimation of glucose uptake in cells, can potentially overcome these problems [6]. One such widely used compound is 2-deoxy-2-(7-Nitro-2,1,3-benzoxadiazol-4-yl)amino-D-glucose (2NBDG), in which the 2-hydroxyl group of D-glucose is replaced with a fluorescent 7-Nitrobenzofurazan group [7]. This compound was first characterized in E. coli where it competed with D-glucose for import via a mannose or a glucose/mannose transporter system [7, 8]. Since its discovery, it has been used across many mammalian cell types and in vitro culture models as a surrogate for glucose uptake. In plasma cells, we used this analog to demonstrate that 2NBDG+ long-lived plasma cells showed elevated spare respiratory capacity relative to their 2NBDG- short-lived counterparts, thereby linking glucose uptake with plasma cell longevity [9, 10]. Further, as compared to other markers of murine plasma cells, 2NBDG positivity correlated well with the longevity of the plasma cell subset [11]. Yet despite its widespread use, 2NBDG uptake has not been definitively shown to occur through glucose transporters.

Glucose import into eukaryotic cells can take place via three families of transporters: the sodium-glucose linked symporters of the SGLT/SLC5 family of transporters; the SWEET family of glucose transporters of the SLC50 family; and lastly, the well characterized GLUT/SLC2 family of sugar transporters [12, 13]. Through much of B cell development and activation, glucose uptake is mediated by the Slc2 family member GLUT1, which is thus likely to be the chief glucose transporter in plasma cells [14]. Our assumption had been that GLUT1 would be the likeliest candidate transporter for 2NBDG uptake in plasma cells as well. Contrary to our expectations, we demonstrate in this study that disruption of GLUT1 expression led to loss of glucose import but had no effect on 2NBDG uptake. Ablation of other candidate transporters also did not affect 2NBDG uptake. These data are consistent with three very recent pharmacological and genetic studies [15–17]. We conclude that 2NBDG is transported independently of known glucose transporters and thus should not be used to estimate glucose uptake by mammalian cells.

Materials and methods

Ethics statement

All animal procedures carried out in this manuscript were approved and carried out based on guidelines provided by the Institutional Animal Care and Use committee at The University of Arizona (approval 17–266). Euthanasia was performed by administering carbon dioxide at a rate of 1.5L/minute in a 7L chamber until 1 minute after respiration ceased. Mice were then cervically dislocated to ensure death.

Mice

C57BL/6N mice were purchased from the Charles River laboratories and housed under specific pathogen free conditions. Experiments were carried out on sex-matched mice between 8–12 weeks of age.

Primary cells and cell lines

The mouse myeloma line 5TGM1 was a gift from Michael H. Tomasson at the Washington University in St. Louis [18]. They were maintained in a T-25 flask at 37°C with 5% CO₂ and split every 4 days at a ratio of 1:10. Cas9-expressing 5TGM1 cells were generated by spin infecting 2x10⁶ 5TGM1 cells with lentiCas9-BLAST lentivirus and 8μg/mL of Polybrene (Millipore Sigma) at 2500rpm for 90 minutes followed by selection in 10μg/mL Blasticidin-S-HCl (Gibco). Guide RNA (gRNA) containing lentiviruses were introduced into these cells by similar spin infections, followed by selection with 10μg/mL Puromycin (Gibco) at 48 hours. Alternatively, 5TGM1 cells were spin-infected with lentiCRISPRv2-mCherry lentivirus to generate a Cas9-expressing line that showed mCherry fluorescence. These cells were purified from uninfected cells by fluorescence-activated cell sorting. LentiX-293T cells (632180, Takara Bio) were used for lentivirus packaging and assembly. Cells were cultured in a 100mm petri dish and split using 0.05% Trypsin-EDTA (Millipore Sigma) when it exhibited greater than 80% confluence. Single cell suspensions from spleens and bone marrows of mice were prepared and erythrocytes lysed with an ammonium chloride-potassium (ACK) lysis buffer. Lymphocytes were then isolated using a Histopaque-1119 (Millipore Sigma) and cells were suspended in 1x PBS containing 5% adult bovine serum (FACS buffer).

Chemicals and cell culture media

2NBDG (11046) and 1NBDF (9002314) were both purchased from Cayman chemical. These reagents were resuspended at a final concentration of 10mg/mL in 1xPBS and in some cases diluted further to 1mg/mL in 1xPBS. Both reagents were completely soluble at both concentrations and did not require a solubilizing agent. Mice were injected intravenously with 100μg of either 2NBDG or 1NBDF and euthanized after 20 minutes. To measure 2NBDG uptake in vitro, 1x10⁶ 5TGM1 cells were cultured with 20μg/mL (~60μM) 2NBDG (Cayman Chemical) in complete media for 1 hour at 37°C followed by antibody staining unless otherwise indicated. 5TGM1 cells were cultured in RPMI (Gibco) containing 10% fetal bovine serum (PEAK Serum), 2mM L-alanyl-L-glutamine, 1mM sodium pyruvate, minimal non-essential amino acids, penicillin, and streptomycin (all from Corning). Glucose-free RPMI (11879–020, Gibco) was used for some experiments, and extraneous D-glucose (G5767, Millipore Sigma) was supplemented to the media at the indicated final concentrations. Lenti-X 293T cells were maintained in DMEM (11965, Gibco) containing 10% fetal bovine serum, L-alanyl-L-glutamine, minimal non-essential amino acids, sodium pyruvate, penicillin, and streptomycin. For assays in sodium-free media, minimum essential medium was used in which sodium salts were replaced by an equal quantity of potassium salts. GLUT1 inhibitors cytochalasin B, BAY-876, and WZB-117 (C6762, SML1774, and SML0621 respectively, all from Millipore Sigma) were all dissolved in DMSO and used at their indicated concentrations. Carbenoxolone (C4790, Millipore Sigma) was dissolved in sterile water and diluted to its final concentration in complete RPMI. For some experiments, 4-Chloro-7-Nitrobenzofurazan (A14165, Thermo Fisher Scientific) was diluted in methanol and used.

Plasmids and lentivirus generation

lentiCas9-Blast, lentiCRISPRv2-mCherry, and lentiGuide-puro were all used in this study (52962, 52961, and 52963 respectively; Addgene). gRNA sequences were chosen from the existing mouse Brie library or designed using the CRISPick platform and cloned into lentiGuide-puro as described previously [19–21]. In brief, 20-mer gRNA sequences were introduced into primers (Millipore Sigma) and after hybridization were cloned into Esp3I-digested lentiGuide-puro using the NEBuilder® HiFi DNA Assembly Master mix (E2621, NEB). Successful constructs were verified in plasmid from isolated colonies by sequencing with the LKO.1 5’ primer. Constructs used for generation of the Slc2a3, Slc2a5, Slc2a6, and Slc2a8 knockout cell line were prepared by digesting lentiGuide-puro constructs with already cloned gRNA sequences and control lentiGuide-puro with BsiWI-HF and MluI-HF (R0553 and R3198, NEB) to exclude the puromycin N-acetyltransferase gene. Sequences encoding fluorescent proteins were then cloned into these digested vectors to generate Slc2a3-lentiGuide-dsRed, Slc2a5-lentiGuide-mMaroon, Slc2a6-lentiGuide-mCherry, and Slc2a8-lentiGuide-tagBFP along with their respective no gRNA controls.

LentiX-293T cells (632180, Takara Bio) were cultured to 60% confluence in 10cm² dishes and transfected using 30μL GeneJuice Transfection reagent (Millipore Sigma), 1.75μg of pMD2.G (12259, Addgene), 3.25μg of psPAX2 (12260, Addgene), and 5μg of the lentiviral construct as per the manufacturer’s instructions. Media was changed at 6 hours post transfection and supernatants collected at 48 and 72 hours and filtered through a 0.45μm syringe. Filtered supernatants were then mixed in a 5:1 ratio with 25% polyethylene glycol-8000 (Millipore Sigma) in 1xPBS and incubated overnight at 4°C. Samples were then spun down at 2500rpm for 20 minutes and pellets resuspended in 100μL 1xPBS. Aliquots were then frozen at -80°C until use.

Flow cytometry

All fluorescence associated cell sorting was carried out on a FACS Aria II and analysis on a BD LSR II or a BD Fortessa (Becton Dickinson). The following anti-mouse antibodies were used: CD138-Phycoerythrin (PE), -Allophycocyanin (APC), or -BV510 (281–2, Biolegend) and B220-BV421 (RA3-6B2, Biolegend). When staining cultured cells, propidium iodide (Millipore Sigma) or Zombie UV (Biolegend) were used to exclude dead cells from the analysis. To stain for GLUT1, cells were first fixed in 2% paraformaldehyde (Electron Microscopy Services), permeabilized in 0.1% Saponin (84510, Millipore Sigma) and then stained with an unconjugated GLUT1 monoclonal antibody (SPM498, Thermo Fisher Scientific) followed by a Rat anti-mouse IgG2a -Alexa Fluor 647 detection antibody (SB84a, Southern Biotech). Cells stained with the detection antibody alone were used as an isotype control. Data was analyzed using the FlowJo software (Becton Dickinson). To monitor 2NBDG uptake kinetics, 1x10⁶ cells were resuspended in the appropriate buffer and 2NBDG introduced to the suspension just prior to recording the sample. Mean Fluorescence Intensity (MFI) changes over time were monitored using the Kinetics platform on FlowJo.

Imaging flow cytometry

2NBDG treated 5TGM1 cells expressing mCherry with control or Slc2a1-targeting gRNA were stained for surface CD138. Samples were then analyzed on an Imagestream^X Mk II (Luminex) and 3000 events recorded per group at 60x magnification. In separate experiments, expression of GLUT1 in deleted cultures were verified by staining cells with CD138 and GLUT1 as described in the previous section. Hoechst 33342 (62249, Thermo Fisher Scientific) was used to identify the nucleus in some assays. Raw information files analyzed on the IDEAS v.6.3 software (Luminex) and similarity morphology indices calculated using the nuclear localization wizard. Mean similarity morphology indices for groups across three experiments were then graphed using Prism 9.2 (Graphpad).

¹⁴C-Glucose uptake assays

1x10⁶ 5TGM1 cells were resuspended in glucose-free incomplete RPMI (Gibco) in a microcentrifuge tube and incubated briefly in a water bath set at 37°C. ¹⁴C Glucose (275 mCi/mmol; NEC042X050UC, Perkin Elmer) was introduced into these cultures to reach a final concentration of 0.5 μCi/mL and cells were allowed to incubate with shaking for 30 minutes. Cells were then washed once with 1x PBS (HyClone) and then lysed in a solution containing freshly prepared 0.5N NaOH and 1% SDS for 30 minutes at room temperature. After neutralization with 1N HCl, cell lysate was loaded onto a 96-well plate (Grenier-BioOne) and mixed with MICROSCINT^TM-20 scintillation fluid (Perkin Elmer). Samples were then incubated at room temperature for 2 hours and incorporated radioactivity analyzed on a 1450 MicroBeta TriLux Microplate Scintillation and Luminescence counter (Perkin Elmer).

Next generation sequencing

Genomic DNA was isolated from gRNA-transduced cultures at the time of assay using a DNA extraction kit (IBI Scientific). Primers were designed +/-150bp from the PAM site of the targeting gRNA and used to amplify 300bp amplicons from 1μg genomic DNA using the Q5® DNA polymerase (M0491, NEB) for 25 cycles. Amplicons were then gel extracted, amplified with the same primers and reaction conditions for 2 cycles, and purified by gel extraction. Equimolar concentrations of amplicons from different reactions were then pooled and 300fmol of the sample was end-prep treated and ligated with adapter sequences according to the ligation sequencing kit protocol (SQK-LSK109, Oxford Nanopore). For some libraries, barcodes were introduced into the samples using a modified version of the ligation sequencing kit protocol (NBD-104, Oxford Nanopore). Samples were then loaded onto primed SpotON flow cells and sequenced on a MinION Mk1c (Oxford Nanopore) with a read filter of 250-500bp and high-accuracy basecalling. Reads in resultant FASTQ files were mapped to the region of interest and indels calculated using the CRISPResso2 analysis pipeline [22]. Unmodified, in-frame, and frame-shift mutation containing read frequencies were graphed as stacked columns using Prism 9.2 (Graphpad).

RNA sequencing

Total RNA was extracted from control gRNA-, Slc2a1 gRNA-, or Slc2a1/3/5/6/8 gRNA-transduced 5TGM1-Cas9 cells using the Nucleospin RNA XS kit (Takara) according to the manufacturer’s instructions. cDNA was generated using oligo-dT primers, fragmented, and Illumina primers ligated for sequencing by the Novogene Corporation Inc. Paired 150bp reads were then sequenced on a NovaSeq 6000 (Illumina). FASTQ files containing 48–69 million reads were then mapped using Salmon and differential gene expression analysis carried out using DESeq2 [23, 24]. All analysis was done by uploading FASTQ files onto the Galaxy public platform, https://usegalaxy.org [25]. Data was plotted on Prism 9.2 (Graphpad). The accession number for the RNA-seq data reported in this paper is NCBI GEO GSE202181.

Statistical analysis

All statistical analysis was carried out on Prism 9.2 (Graphpad). Specific tests used and significance are indicated in the figures and accompanying figure legends. Adjusted p-values and fold changes for RNA-Seq data were calculated on DESeq2 [24].

Results

GLUT1 does not mediate uptake of 2NBDG

The glucose transporter GLUT1, encoded by the gene Slc2a1, is highly expressed by multiple myeloma cells, which are transformed counterparts of long-lived plasma cells [26]. Using lentiviral transduction, we engineered 5TGM1 mouse myeloma cells to constitutively express the Cas9 protein. Using lentiviruses, we then expressed four different guide RNAs (gRNAs) that targeted exons 3, 4, or 5 in the Slc2a1 locus in these 5TGM1-Cas9 cells. The frequency of GLUT1-positive cells in these cultures dropped to approximately 50% relative to that of a control gRNA-transduced culture (Fig 1A). Consistent with a previous report, we observed GLUT1 at the plasma membrane as determined by colocalization with the surface marker CD138 in GLUT1-sufficient cells(S1 Fig) [27]. As expected, GLUT1-negative cells in these same Slc2a1-deleted cultures, however, showed complete loss of this transporter on both the surface and in the cytosol (S1 Fig). This loss in expression of GLUT1 was accompanied by an approximate 80% decrease in ¹⁴C-glucose uptake (Fig 1B). Some of the GLUT1-positive cells in the deleted cultures may represent cells with one functional copy of GLUT1 and as a result show a partial reduction in ¹⁴C-glucose uptake. Further, ¹⁴C-glucose can be catabolized into various cellular metabolites, indicating a simultaneous measure of uptake and metabolism in this assay. Put together, this might explain the slight discrepancy between the degree of reduction in GLUT1-positive cells and the extent of ¹⁴C-glucose uptake in deleted cultures. To our surprise, however, 2NBDG import was unaffected in Slc2a1-targeted gRNA cultures (Fig 1C). Further, we saw no change in the kinetics of 2NBDG uptake in these cells, measured over the course of an hour (Fig 1D). These findings suggest that mechanisms or transporters other than GLUT1 can mediate uptake of 2NBDG.

Fig 1 — (A) GLUT1 staining of gRNA-transduced cells. Representative histogram (left) showing GLUT1 expression on 5TGM1 cells transduced with a control gRNA (black) and a *Slc2a1*-targeting gRNA (blue). Quantification of percent GLUT1 positive cells (right) in control and *Slc2a1* gRNA-transduced cells across 9 independent experiments. Each circle represents a single group from a single experiment. ***p<0.0001 by Brown-Forsyth and Welch one-way ANOVA multiple comparisons test. (B) ¹⁴C-glucose uptake in gRNA-transduced cells. Lysates from cells transduced with gRNAs as in (A) were cultured in ¹⁴C-glucose containing media for 30 minutes and ¹⁴C signal was quantified in the cell pellet. Background counts indicated by the dotted line. Representative graph of three independent experiments. *p<0.05 by Brown-Forsyth and Welch one-way ANOVA multiple comparisons test. (C) Flow cytometric analysis of 2NBDG uptake in control gRNA- (black) and *Slc2a1* gRNA-transduced (colored) 5TGM1-Cas9 cells. Cells were cultured in media containing 2NBDG for 60 minutes. Histogram representative of three independent experiments. (D) 2NBDG uptake in control gRNA (black) and *Slc2a1* gRNA-transduced (colored) 5TGM1-Cas9 cells. Mean fluorescence intensity (MFI) +/- SEM shown for 0-, 15-, 30-, 45-, and 60-minutes post culturing with 2NBDG. Pooled data from three independent experiments. No significant differences were observed with ordinary two-way ANOVA with Dunnett’s multiple comparison test.

Plasma cells take up 2NBDG, but not 1-NBD-Fructose

We next explored other possible mechanisms by which cells take up 2NBDG. Solute entry into cells can take place via three main pathways: diffusion, endocytosis-mediated uptake, or import through solute carriers and other transporters. We considered the possibility that 2NBDG, which is hydrophilic, is endocytosed by cells in a non-specific manner rather than delivered to the cytoplasm via a transporter. To test this, we examined 5TGM1 cells treated with 2NBDG using imaging flow cytometry and observed that 2NBDG was distributed evenly across the cytosol of cells (Fig 2A). Weak colocalization was observed with the nuclear stain Hoechst 33342 and no colocalization with the surface marker CD138 (Fig 2A). No punctate localization was observed as one would expect from vesicle-mediated uptake or endocytosis (Fig 2A). The absence of the sugar transporter GLUT1 did not affect this distribution, confirming a Slc2a1-independent mechanism of 2NBDG uptake (Fig 2B).

Fig 2 — (A) 2NBDG is retained in the cytosol of cells. 5TGM1 cells previously transduced with a Cas9-T2A-mCherry lentivirus were treated with 2NBDG, stained for surface CD138 and Hoeschst 33342, and analyzed by imaging flow cytometry. Representative images of cells treated with 2NBDG (top left) relative to untreated cells (bottom left) at 60x magnification are shown. Mean similarity morphology indices for 2NBDG and other stains/dyes were quantified and shown (right). (B) Cas9-mCherry expressing 5TGM1 cells were transduced with control gRNA or *Slc2a1* gRNA lentiviruses. Representative images of each gRNA group (left) at 60x magnification are shown with overlays for 2NBDG/CD138 and 2NBDG/mCherry. Mean similarity morphology indices were quantified (right). For both panels, each dot indicates the mean value for a group in a single experiment. No significant differences were observed with ordinary two-way ANOVA with Dunnett’s multiple comparison test.

We next examined the specificity of 2NBDG uptake in plasma cells. We injected mice with 2NBDG or 1-NBD-fructose (1NBDF), a derivative of fructose in which the NBD moiety is attached to the C-1 sugar of fructose (S2 Fig) [28]. We observed 2NBDG uptake in both splenic and bone marrow plasma cells, with the latter showing a higher frequency of 2NBDG-positive cells (Fig 3A), as we previously reported [9, 10]. Plasma cells, however, did not detectably import 1NBDF (Fig 3A). These data argue against endocytic fluid phase accumulation of 2NBDG, as this mechanism would be predicted to also lead to the accumulation of the chemically similar 1NBDF. We also measured uptake of 4-Chloro-7-nitrobenzofurazan (4C7NB), the fluorescent substrate used in the synthesis of 2NBDG. Unlike 2NBDG, this compound is hydrophobic and showed a linear increase in MFI over time in 5TGM1 cells (S3 Fig). 5TGM1 cells treated with 2NBDG, however, reached a steady state intensity in less than 30 seconds and maintained it for the remainder of the assay (S3 Fig). This pattern of kinetics of 2NBDG accumulation is indicative of transporter-mediated uptake, as under conditions of diffusion or endocytosis, uptake is a linear function of time. We observed similar 2NBDG uptake kinetics in primary mouse plasma cells, suggesting that the mechanism of 2NBDG transport is similar between the two cell types (Fig 3B). Further, 2NBDG import was higher in plasma cells as compared to B cells and total spleen cells (Fig 3B). Put together, these data suggest that plasma cells specifically import 2NBDG through a transporter(s) other than GLUT1.

Fig 3 — (A) Mice were injected with 100μg of either 2NBDG or 1NBDF and assessed for NB fluorescence in splenic and bone marrow plasma cells. Representative flow cytometry plots (left) on splenic (top row) and bone marrow (bottom row) CD138+ plasma cells showing gated percent NB-positive cells. Quantification of NB-positive plasma cell percentages (left) in the spleen and bone marrow and groups from each mouse are connected by a line. Data from three independent experiments with n = 8 mice in both groups. ****p<0.0001 by ordinary two-way ANOVA with post hoc Šídák’s multiple comparison test. (B) Freshly isolated *ex vivo* plasma cells (CD138+ B220+/-, orange), B cells (CD138- B220+, purple), and total spleen cells (green) as well as cultured 5TGM1 cells (black) were examined for 2NBDG uptake by flow cytometry. Pooled data from three independent experiments shown as mean+/-SEM for mentioned time points.

GLUT1 inhibition or ablation does not affect 2NBDG uptake kinetics

To further assess the role of GLUT1 in 2NBDG uptake, we pharmacologically inhibited GLUT1 by treating cells with cytochalasin B (CytoB), a well-established GLUT1 inhibitor [29, 30]. As CytoB also affects actin polymerization, we tested 2NBDG uptake in the presence of more specific GLUT1 inhibitors, namely BAY-876 and WZB-117 [31–33]. We found no change in 2NBDG intensity in drug-treated groups compared to untreated or DMSO-treated cells (Fig 4A). We also tested the role of GAP junctions and hemidesmosomes in 2NBDG import by treating cells with the blocker Carbenoxolone (CBX) [33–35]. We found no effect of the compound on 2NBDG uptake (Fig 4B). Further, genetic ablation of Slc2a1 in 5TGM1 cells did not affect 2NBDG import in 5TGM1 cells at any of the time points assayed (Fig 4C). Put together, these findings strongly suggest that 2NBDG transport is independent of GLUT1 and glucose uptake.

Fig 4 — (A) 5TGM1 cells were treated with DMSO, 30μM Cytochalasin B (CytoB), 10μM BAY-876, or 100μM WZB-117 for 30 minutes in complete media followed by 60μM of 2NBDG for another 30 minutes. Mean +/- SEM of 2NBDG MFI are shown as bar graphs. Pooled data from three independent experiments. No significance was observed by one-way ANOVA. (B) 5TGM1 cells were treated with 300nM Carbenoxolone (CBX) and assayed for 2NBDG uptake as in (A). Mean +/- SEM of 2NBDG MFI are shown as bar graphs. Pooled data from three independent experiments. No significance observed by the paired t-test. (C) Control gRNA- or *Slc2a1* gRNA-transduced 5TGM1-Cas9 cells were administered 2NBDG at a final concentration of 60μM and intensity monitored immediately after addition by flow cytometry. Mean +/- SEM is shown for each group for the mentioned time points. Pooled data from three independent experiments.

2NBDG uptake does not depend on other glucose transporters

We next hypothesized that 2NBDG uptake into cells could take place via other transporters implicated in glucose uptake. We performed RNA-Seq on 5TGM1 cells and compared the gene expression profiles with published gene expression data on 5TGM1 cells and primary plasma cells to identify candidate glucose and 2NBDG transporters of the Slc2, Slc5, and Slc50 families [9, 10, 36]. Of the 13 members of the Slc2 family in mice, primary plasma cells and/or myeloma cells expressed SLC2A3, SLC2A6, and SLC2A8 as candidate glucose transporters in addition to SLC2A1 (Fig 5A). Data from another group also identified SLC2A5 in addition to the aforementioned SLC2 transporters [36]. Expression of SLC5 family members was not detected. We also observed high levels of expression of SLC50A1, though this transporter is primarily involved in glucose efflux rather than import [13].

Fig 5 — (A) Transcripts per million kilobase (TPM) values of sugar transporters in *ex vivo* bone marrow plasma cells (orange) and 5TGM1 cells (black). Mean values +/- SEM are shown. (B) Quantification of gene modifications in gRNA-transduced cultures. Exons of indicated genes were PCR amplified and sequenced to assay for in-frame and frame shift mutations. Mean values +/- SEM shown for each of the genes and modifications within it. Pooled data from three experiments. (C) 2NBDG uptake in sugar transporter deleted cultures. 5TGM1-Cas9 cells were transduced with control gRNA (black) or gRNAs targeting *Slc2a3* (pink), *Slc2a5* (blue), *Slc2a6* (violet), *Slc2a8* (purple), and *Slc50a1* (cyan). MFIs across three independent experiments are quantified and displayed with SEM. No significant differences observed with Brown-Forsyth and Welch one-way ANOVA multiple comparisons test. (D) Frequency of 2NBDG-negative cells for groups in (C). 2NBDG- gate drawn based on control cells cultured without 2NBDG in complete media. Mean values +/- SEM are shown. No significant differences observed with Brown-Forsyth and Welch one-way ANOVA multiple comparisons test.

To test if these transporters are responsible for 2NBDG import, we transduced gRNAs targeting these genes into 5TGM1-Cas9 cells and measured 2NBDG uptake by flow cytometry. Sequencing of gRNA targets in these cultures demonstrated 31–50% frameshift mutations, confirming efficient ablation of the intended transporters (Fig 5B). Disruption of these genes, however, did not affect 2NBDG uptake (Fig 5C). Moreover, the frequency of 2NBDG-negative cells in all cultures was similar to those seen in control gRNA-transduced cultures (Fig 5D).

One possible explanation for the unaltered 2NBDG uptake in the Slc2a1, Slc2a3, Slc2a5, Slc2a6, and Slc2a8 deleted cultures is functional redundancy between these sugar transporters. As a result, loss of one of these genes might be compensated by activity of other transporters. To test this possibility, we generated 5TGM1 cells carrying gRNAs targeting all the expressed members of the SLC2 family. Because GLUT1-deficient cells show reduced viability, we first generated a cell line that was ablated for Slc2a3, Slc2a5, Slc2a6, and Slc2a8 using lentiviral transduction followed by fluorescence activated cell sorting of the reporter positive cells. After sorting and expansion of this line, we then introduced lentiviruses expressing a gRNA targeting Slc2a1 and examined for 2NBDG uptake 4 days after transduction. Analysis of Slc2a1 sequences in these cultures showed an average of 50% frameshift mutations, while the other Slc2 members had 54–65% frameshifts in their respective sequences (Fig 6A). Yet 2NBDG uptake was equivalent in these cells relative to controls (Fig 6B–6D).

Fig 6 — (A) Quantification of in-frame and frameshift mutations in *Slc2a1*, *Slc2a3*, *Slc2a5*, *Slc2a6*, and *Slc2a8* genes of the *Slc2a1/3/5/6/8* deleted cultures. Pooled data from three experiments. (B) 2NBDG uptake in *Slc2a1/3/5/6/8* cultures. Representative histogram (left) showing control gRNA- (black) and the five gRNA-transduced cultures (green). Pooled MFI +/- SEM for both groups across three independent experiments are depicted (right). No significance observed with the paired t-test. (C) Frequencies of 2NBDG-negative cells in cultures described in (B). 2NBDG- gating carried out as in Fig 5D. Mean values +/- SEM are shown for both groups. No significance observed with the paired t-test.

We considered the possibility that Slc2a1-deleted 5TGM1 cells upregulate other members of the SLC2 family as a compensatory mechanism. Relative to control gRNA-transduced cells, we observe a statistically significant reduction in SLC2A1 and SLC50A1 transcript levels in the deleted cultures, but no noticeable change in the transcript levels of any of the other members of the SLC2 and SLC5 families (S4 Fig). Thus, no known glucose transporter is involved in 2NBDG uptake.

Exogenous glucose does not inhibit 2NBDG uptake

We next hypothesized that 2NBDG uptake may be mediated by an unidentified glucose transporter. In this case, 2NBDG import into cells would be impeded by competing amounts of D-glucose in the culture medium. To test this, we quantified 2NBDG uptake in 5TGM1 cells suspended in glucose-free RPMI-1640 or with media containing increasing amounts of D-glucose, reaching 500-fold excess relative to 2NBDG concentrations in the media. By flow cytometry, we observed 2NBDG uptake to reach a steady state in all groups rapidly post-addition but found no difference in the mean 2NBDG intensity in cells under glucose-sufficient or -deficient conditions (Fig 7A). We observed a similar trend in ex vivo primary plasma cells, where if anything, cells showed higher 2NBDG intensity in the presence of D-glucose (Fig 7B). Competing glucose in the assay media did not impact 2NBDG intensities in splenic B cells or total spleen cells at any point in the assay (Fig 7C and 7D). Put together, these findings indicate that 2NBDG import is independent of glucose uptake and transporters.

Fig 7 — (A) 5TGM1 cells were resuspended in either glucose-free RPMI or RPMI with D-glucose at the mentioned final concentrations. 2NBDG was added at a final concentration of 60μM and intensity monitored immediately after addition by flow cytometry. (B-D) CD138+ enriched spleen cells were resuspended in glucose-free RPMI or RPMI with D-glucose at the specified concentrations. 2NBDG was added and intensity monitored immediately by flow cytometry. 2NBDG intensity in (B) plasma cells (CD138+ B220+/-), (C) B cells (CD138- B220+), and (D) unenriched total spleen cells are shown for the mentioned time points. Mean +/- SEM shown for each group for the indicated time points. Pooled data from three independent experiments each.

Nucleoside and nucleoside-sugar transporters do not transport 2NBDG

Given the utility of 2NBDG import as a marker of plasma cell longevity, we performed experiments to test other candidate transporters. The structure of 2NBDG mimics nucleotides and nucleotide sugars, which are imported through the Slc29 and Slc35 families of transporters, respectively (S2 Fig). RNA-seq data showed Slc29a1 and Slc29a3 expression in both primary bone marrow plasma cells and 5TGM1 cells (Fig 8A). Moreover, 17 of the 27 known Slc35 members in mice were also expressed (Fig 8A). CRISPR-Cas9 ablation of these transporters led to 20–59% frameshift mutations in sequences from 5TGM1 cultures (Fig 8B). However, none of these mutations affected 2NBDG uptake (Fig 8C and 8D). Thus, the Slc29 or Slc35 families of nucleotide and nucleotide sugar transporters are not required for 2NBDG uptake.

Fig 8 — (A) TPM values of nucleoside and nucleoside-sugar transporters in *ex vivo* bone marrow (orange) plasma cells and 5TGM1 cells (black). Mean values +/- SEM are shown. (B) Estimation of indels in nucleoside and nucleoside-sugar transporter deleted cultures done as in Fig 5B. Mean values +/- SEM shown for each of the genes and modifications within it. Pooled data from three independent experiments. (C) 2NBDG uptake in deleted cultures. 5TGM1-Cas9 cells transduced with gRNAs targeting indicated nucleoside or nucleoside-sugar transporters (colored) or with a control gRNA (black). 2NBDG MFIs across three independent experiments are quantified and displayed with SEM. (D) Frequencies of 2NBDG negative cells in cultures described in (C). Pooled data from three experiments. No statistical significance observed with the Brown-Forsyth and Welch one-way ANOVA multiple comparisons test.

2NBDG uptake is a low-affinity, sodium independent process

To further understand the kinetics of 2NBDG uptake in cells, we next examined 2NBDG import in 5TGM1 cells at differing concentrations of 2NBDG. This would enable us to assess saturability of the compound and estimate parameters like the Michaelis-Menten constant (Km) and Vmax, thereby narrowing down a list of candidate transporters. We treated 5TGM1 cells with dilutions of 2NBDG, starting at a maximum concentration of 6mM and titrating down to 6μM, the lowest dose at which we could detect fluorescence above background. We found that 2NBDG steady state intensity was reached rapidly and maximum intensity titrated nearly proportional to the concentration of 2NBDG (Fig 9A). We did not observe saturation at any of the concentrations we tested, thereby precluding calculations for Km and Vmax. We also examined 2NBDG transport in the absence of exogenous sodium ions, an important cofactor for multiple symporter and antiporter systems in the cell, including the SGLT/SLC5 family of transporters [37]. Cells in sodium free media, however, showed a higher 2NBDG intensity at all time points assayed (Fig 9B). We may infer from these findings that 2NBDG uptake is possibly a low affinity process and does not require sodium ions to mediate its uptake. Further, while specific, 2NBDG uptake is not mediated by known sugar, nucleotide, or nucleotide sugar transporters.

Fig 9 — (A) 2NBDG was added to 5TGM1 cells resuspended in 1xPBS to the specified final concentrations and uptake measured by flow cytometry. (B) 5TGM1 cells were resuspended in either DMEM or Sodium-free MEM and measured for 2NBDG import by flow cytometry. Mean +/- SEM shown for each group for the indicated time points. Pooled data from three independent experiments each.

Discussion

Fluorescent derivatives of glucose like 2NBDG have been used as a tool for visualizing glucose uptake in cells and in vivo. However, its import through mammalian sugar transporters has not been demonstrated genetically. Using CRISPR-Cas9 editing of myeloma cells, we show that the sugar transporter GLUT1 is important for glucose uptake but has no role in 2NBDG transport in these cells. Although glucose can be imported into cells via other sugar transporters, we also found no role for these alternative routes in 2NBDG transport. Finally, we found that excess glucose had no impact on 2NBDG uptake in primary splenocytes. As such, our findings show a disconnect between glucose uptake and 2NBDG transport in mammalian cells.

During the course of this work, three reports were published by independent groups that arrived at similar conclusions. The first report by Sinclair et al. demonstrated that double-positive (DP) thymocytes took up very little glucose as compared to activated CD8+ T cells in culture [15]. The 2NBDG uptake by cells in these cultures, however, showed the exact opposite trend and was unaffected by pharmacological inhibition of glucose transporters [15]. The second group took advantage of the L929 fibroblast line which expresses GLUT1 as its sole glucose transporter [38]. Using pharmacological inhibitors, siRNA mediated knockdowns, and GLUT1 overexpression, the authors were able to modulate glucose uptake in these cells, but not of 2NBDG or another fluorescent derivative of glucose, 6NBDG [16]. Another recent study observed a lack of in vivo correlation between uptake of ¹⁸F-fluorodeoxyglucose, a well-verified reporter of glucose uptake, and 2NBDG [17]. Our findings provide an independent genetic confirmation for each of these reports, and further extend the disparity between glucose and 2NBDG uptake to other glucose transporters within the Slc2 and Slc50 families. The use of CRISPR-Cas9 to induce gene deletions in our system offers a robust means to provide this independent confirmation.

In plasma cells, 2NBDG uptake marks longer lived subsets and identifying its transporter could provide insights into mechanisms regulating the longevity of these cells. Long-lived plasma cells do possess more glucose-dependent spare respiratory capacity than do short-lived plasma cells [10]. However, our original conclusions that long-lived plasma cells import more glucose than their short-lived counterparts will need to be revisited. The as-yet unidentified transporter seems specific, as our data indicate that cells deposit 2NBDG in the cytosol. The kinetics of 2NBDG uptake would indicate it is mediated through a transporter, albeit through a low-affinity process. We attempted an unbiased genome-wide gRNA library screen in the 5TGM1 cell line but were unable to identify any known candidates in sorted 2NBDG-negative cultures. The lack of any putative targets in the screen suggests that 2NBDG might be imported into cells via multiple functionally redundant transporters. Such transporters could potentially be identified through an overexpression library screen in cells that intrinsically do not take up 2NBDG. As the mechanisms of 2NBDG uptake remain unclear, we strongly advise that despite its convenience, it should not be considered as a direct indicator of glucose uptake.

Supporting information

S1 Fig. Slc2a1 deletion leads to a loss of surface GLUT1 expression.

Cells transduced with Slc2a1 gRNA were stained for GLUT1 and CD138. (A) Representative images from GLUT1-sufficient (top) and GLUT1-deficient cells (bottom) in the same culture. (B) Quantification of mean similarity morphology indices for GLUT1 and CD138 in GLUT1+ (filled bars) and GLUT1- (hollow bars) cells. Pooled data from three independent experiments. *p<0.05 by Šídák’s multiple comparisons test.

(TIF)

Click here for additional data file.^{(523.9KB, tif)}

S2 Fig. Structures of 2NBDG and 1NBDF in relation to glucose and naturally occurring nucleosides.

Chemical structures of (A) D-Glucose, (B) 2NBDG, (C) 1NBDF, (D) 4C7NB, (E) Guanosine, (F) Adenosine, (G) Deoxythymidine, (H) Uridine, and (I) Cytosine. Structures generated using ChemDraw v.20.1.1.

(TIF)

Click here for additional data file.^{(347.1KB, tif)}

S3 Fig. 2NBDG and 4C7NB show different uptake kinetics in 5TGM1 cells. 5TGM1 cells were treated with 60μM 2NBDG (black) or 1μM 4C7NB (orange) and the intensity of each compound was monitored over time by flow cytometry. Pooled data from three independent experiments.

(TIF)

Click here for additional data file.^{(206.5KB, tif)}

S4 Fig. Slc2 deletion does not lead to compensatory expression of other transporters.

RNA-Seq analysis of sugar transporter transcript levels in control gRNA-transduced (black), Slc2a1 gRNA-transduced (blue), and Slc2a1/3/5/6/8 gRNA-transduced (green) 5TGM1-Cas9 cultures. Three biological replicates were analyzed for each population and data is represented as mean values +/- SEM. *p<0.05, **p<0.01, and ***p<0.001 by paired two-way ANOVA.

(TIF)

Click here for additional data file.^{(310.3KB, tif)}

Acknowledgments

We wish to thank the flow cytometry core at the University of Arizona for their assistance with flow cytometry and the Nikolich-Žugich laboratory for the use of the BD Fortessa for some experiments. We are also grateful to Tyler J. Ripperger for his assistance with RNA-Seq.

Data Availability

RNA-seq data reported in this paper is available at NCBI GEO (accession GSE202181). All other relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by NIH grant R01AI129945 (D.B.). L. D. was supported by a Bio5 Postdoctoral fellowship award. The use of the Imagestream was made possible by the NIH award S10 OD028466. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study.

References

1.Nelson DL, Cox MM. Lehninger Principles of Biochemistry. 7th ed. New York: W. H. Freeman; 2017. [Google Scholar]
2.Lam WY, Bhattacharya D. Metabolic Links between Plasma Cell Survival, Secretion, and Stress. Trends in Immunology. 2018;39: 19–27. doi: 10.1016/j.it.2017.08.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Som P, Atkins HL, Bandoypadhyay D, Fowler JS, MacGregor RR, Matsui K, et al. A Fluorinated Glucose Analog, 2-fluoro-2-deoxy-D-glucose (F-18): Nontoxic Tracer for Rapid Tumor Detection. Journal of Nuclear Medicine. 1980;21: 670–675. [PubMed] [Google Scholar]
4.Yamamoto N, Ueda-Wakagi M, Sato T, Kawasaki K, Sawada K, Kawabata K, et al. Measurement of Glucose Uptake in Cultured Cells. Current Protocols in Pharmacology. 2015;71: 12.14.1–12.14.26. doi: 10.1002/0471141755.ph1214s71 [DOI] [PubMed] [Google Scholar]
5.Maric T, Mikhaylov G, Khodakivskyi P, Bazhin A, Sinisi R, Bonhoure N, et al. Bioluminescent-based imaging and quantification of glucose uptake in vivo. Nat Methods. 2019;16: 526–532. doi: 10.1038/s41592-019-0421-z [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Kim WH, Lee J, Jung D-W, Williams DR. Visualizing Sweetness: Increasingly Diverse Applications for Fluorescent-Tagged Glucose Bioprobes and Their Recent Structural Modifications. Sensors. 2012;12: 5005–5027. doi: 10.3390/s120405005 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Yoshioka K, Takahashi H, Homma T, Saito M, Oh KB, Nemoto Y, et al. A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli. Biochim Biophys Acta. 1996;1289: 5–9. doi: 10.1016/0304-4165(95)00153-0 [DOI] [PubMed] [Google Scholar]
8.Tao J, Diaz RK, Teixeira CRV, Hackmann TJ. Transport of a Fluorescent Analogue of Glucose (2-NBDG) versus Radiolabeled Sugars by Rumen Bacteria and Escherichia coli. Biochemistry. 2016;55: 2578–2589. doi: 10.1021/acs.biochem.5b01286 [DOI] [PubMed] [Google Scholar]
9.Lam WY, Jash A, Yao C-H, D’Souza L, Wong R, Nunley RM, et al. Metabolic and Transcriptional Modules Independently Diversify Plasma Cell Lifespan and Function. Cell Rep. 2018;24: 2479–2492.e6. doi: 10.1016/j.celrep.2018.07.084 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Lam WY, Becker AM, Kennerly KM, Wong R, Curtis JD, Llufrio EM, et al. Mitochondrial Pyruvate Import Promotes Long-Term Survival of Antibody-Secreting Plasma Cells. Immunity. 2016;45: 60–73. doi: 10.1016/j.immuni.2016.06.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.D’Souza L, Bhattacharya D. Plasma cells: You are what you eat. Immunological Reviews. 2019;288: 161–177. doi: 10.1111/imr.12732 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Deng D, Yan N. GLUT, SGLT, and SWEET: Structural and mechanistic investigations of the glucose transporters. Protein Science. 2016;25: 546–558. doi: 10.1002/pro.2858 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Chen L-Q, Hou B-H, Lalonde S, Takanaga H, Hartung ML, Qu X-Q, et al. Sugar transporters for intercellular exchange and nutrition of pathogens. Nature. 2010;468: 527–532. doi: 10.1038/nature09606 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Caro-Maldonado A, Wang R, Nichols AG, Kuraoka M, Milasta S, Sun LD, et al. Metabolic reprogramming is required for antibody production that is suppressed in anergic but exaggerated in chronically BAFF-exposed B cells. J Immunol. 2014;192: 3626–3636. doi: 10.4049/jimmunol.1302062 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Sinclair LV, Barthelemy C, Cantrell DA. Single Cell Glucose Uptake Assays: A Cautionary Tale. Immunometabolism. 2020;2: e200029. doi: 10.20900/immunometab20200029 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Hamilton KE, Bouwer MF, Louters LL, Looyenga BD. Cellular binding and uptake of fluorescent glucose analogs 2-NBDG and 6-NBDG occurs independent of membrane glucose transporters. Biochimie. 2021;190: 1–11. doi: 10.1016/j.biochi.2021.06.017 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Reinfeld BI, Madden MZ, Wolf MM, Chytil A, Bader JE, Patterson AR, et al. Cell-programmed nutrient partitioning in the tumour microenvironment. Nature. 2021;593: 282–288. doi: 10.1038/s41586-021-03442-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Garrett IR, Dallas S, Radl J, Mundy GR. A murine model of human myeloma bone disease. Bone. 1997;20: 515–520. doi: 10.1016/s8756-3282(97)00056-2 [DOI] [PubMed] [Google Scholar]
19.Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-Guided Human Genome Engineering via Cas9. Science. 2013;339: 823–826. doi: 10.1126/science.1232033 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Sanson KR, Hanna RE, Hegde M, Donovan KF, Strand C, Sullender ME, et al. Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun. 2018;9: 5416. doi: 10.1038/s41467-018-07901-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34: 184–191. doi: 10.1038/nbt.3437 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Clement K, Rees H, Canver MC, Gehrke JM, Farouni R, Hsu JY, et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol. 2019;37: 224–226. doi: 10.1038/s41587-019-0032-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017;14: 417–419. doi: 10.1038/nmeth.4197 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15: 550. doi: 10.1186/s13059-014-0550-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Afgan E, Baker D, van den Beek M, Blankenberg D, Bouvier D, Čech M, et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Research. 2016;44: W3–W10. doi: 10.1093/nar/gkw343 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Wardell SE, Ilkayeva OR, Wieman HL, Frigo DE, Rathmell JC, Newgard CB, et al. Glucose Metabolism as a Target of Histone Deacetylase Inhibitors. Molecular Endocrinology. 2009;23: 388–401. doi: 10.1210/me.2008-0179 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Ogorevc M, Strikic A, Tomas SZ. Determining the immunohistochemical expression of GLUT1 in renal cell carcinoma using the HSCORE method. Biomed Rep. 2021;15: 79. doi: 10.3892/br.2021.1455 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Levi J, Cheng Z, Gheysens O, Patel M, Chan CT, Wang Y, et al. Fluorescent fructose derivatives for imaging breast cancer cells. Bioconjug Chem. 2007;18: 628–634. doi: 10.1021/bc060184s [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Devés R, Krupka RM. Cytochalasin B and the kinetics of inhibition of biological transport. A case of asymmetric binding to the glucose carrier. Biochimica et Biophysica Acta (BBA)—Biomembranes. 1978;510: 339–348. doi: 10.1016/0005-2736(78)90034-2 [DOI] [PubMed] [Google Scholar]
30.Mizel SB, Wilson L. Inhibition of the Transport of Several Hexoses in Mammalian Cells by Cytochalasin B. Journal of Biological Chemistry. 1972;247: 4102–4105. doi: 10.1016/S0021-9258(19)45146-6 [DOI] [PubMed] [Google Scholar]
31.MacLean-Fletcher S, Pollard TD. Mechanism of action of cytochalasin B on actin. Cell. 1980;20: 329–341. doi: 10.1016/0092-8674(80)90619-4 [DOI] [PubMed] [Google Scholar]
32.Siebeneicher H, Cleve A, Rehwinkel H, Neuhaus R, Heisler I, Müller T, et al. Identification and Optimization of the First Highly Selective GLUT1 Inhibitor BAY-876. ChemMedChem. 2016;11: 2261–2271. doi: 10.1002/cmdc.201600276 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Ojelabi OA, Lloyd KP, Simon AH, De Zutter JK, Carruthers A. WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site*. Journal of Biological Chemistry. 2016;291: 26762–26772. doi: 10.1074/jbc.M116.759175 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Davidson JS, Baumgarten IM, Harley EH. Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid. Biochemical and Biophysical Research Communications. 1986;134: 29–36. doi: 10.1016/0006-291x(86)90522-x [DOI] [PubMed] [Google Scholar]
35.Rouach N, Koulakoff A, Abudara V, Willecke K, Giaume C. Astroglial Metabolic Networks Sustain Hippocampal Synaptic Transmission. Science. 2008;322: 1551–1555. doi: 10.1126/science.1164022 [DOI] [PubMed] [Google Scholar]
36.Trotter TN, Li M, Pan Q, Peker D, Rowan PD, Li J, et al. Myeloma cell-derived Runx2 promotes myeloma progression in bone. Blood. 2015;125: 3598–3608. doi: 10.1182/blood-2014-12-613968 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Wright EM, Turk E. The sodium/glucose cotransport family SLC5. Pflugers Arch—Eur J Physiol. 2004;447: 510–518. doi: 10.1007/s00424-003-1063-6 [DOI] [PubMed] [Google Scholar]
38.Liong E, Kong SK, Au KK, Li JY, Xu GY, Lee YL, et al. Inhibition of glucose uptake and suppression of glucose transporter 1 mRNA expression in L929 cells by tumour necrosis factor-α. Life Sciences. 1999;65: PL215–PL220. doi: 10.1016/S0024-3205(99)00408-7 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0261801.r001

Decision Letter 0

Hodaka Fujii

8 Feb 2022

PONE-D-21-38751Genetic evidence that uptake of the fluorescent analog 2NBDG occurs independently of known glucose transportersPLOS ONE

Dear Dr. Bhattacharya,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Your paper was reviewed by four experts. As they point out, your claim is very strong and needs more data to support it. For example, use of other cell types and kinetic analyses must be performed. Please revise your manuscript according to the comments by the reviewers and carefully specify your claims / conclusions based on the data in your hand.

Please submit your revised manuscript by Mar 25 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Hodaka Fujii, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating in your Funding Statement:

(This work was supported by NIH grant R01AI129945 (D.B.). L. D. was supported by a Bio5 Postdoctoral fellowship award. The use of the Imagestream was made possible by the NIH award S10 OD028466. he funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript)

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this paper, the authors present data regarding the effect of CRISPR-mediated deletion of several classes of membrane transport proteins on 2NBDG uptake in primary plasma and 5TGM1 myeloma cell lines. The authors observe continued 2NBDG uptake despite significant reduction in glucose transport with knockdown of the primary GLUT transporter (SLC2A1) in these cells. To search for possible explanations for this unexpected finding, they perform a fairly extensive, though not exhaustive, set of experiments to knock down other transporters, including one experiment where they simultaneously knock down each of the GLUT isoforms detected in the 5TGM3 cells. While overall the data agree with recent reports using other methodologies that 2NBDG may not serve as a reliable indicator of overall glucose uptake and utilization in mammalian cells, this manuscript has several limitations, based largely on the tools utilized and the experimental conditions chosen, that require either modification of the conclusions reached or addition of additional data. In particular, while the identity of the novel putative transporter(s) may not be clear, within the context of this paper it can and should be directly established whether or not sustained 2NBDG import is due to a carrier-mediated process.

Specific comments and critique:

1. In the paper abstract, the authors claim that CRISP-mediated knockout of Slc2a1 alters the kinetics of 2NBDG uptake. However, the paper does not directly assess transport kinetics. The only data shown is a time course for mean fluorescence intensity. The scale shown is relatively long and does not allow assessment of zero-trans uptake. To make this claim, much more data needs to be shown including classic experiments to assess for transport-mediated uptake. This includes demonstration of saturability and at a minimum estimation of the Km and Vmax of the transport process.

2. In Figure 1, there is a discrepancy in the degree of reduction of GLUT1 positive cells (~50%) and the degree of 14C glucose uptake (~80%) that is not adequately addressed in the manuscript text. This may be due in part to the experimental conditions used to assess for glucose transport activity. What is shown is the combined effect in GLUT1 expressing and non-expression cells with 30-minute incubation with radiolabeled substrate. Under these conditions, there are influences of both uptake and metabolism of glucose. It is recommended that assays be performed at shorter time points and with non-metabolizable substrate (e.g. 2-deoxyglucose). At a minimum, the authors need to show (or adequately reference) the kinetics of glucose transport activity in these cell lines under the conditions used.

3. The interpretation of the data shown in Figure 2 is overstated. In these experiments, the authors seek to establish that the uptake of 2NBDG does not occur via a non-specific endocytosis-mediated process. Demonstration of uniform cytosolic staining of 2NBDG does not prove that this is mediated through a transport-mediated process. Furthermore, the comparison to 1NBDF, while showing that there is specificity for 2NBDG over the other substrate (implying an effect on glucose over fructose uptake), it does not directly follow that these data is not from GLUT1 mediated transport.

4. Although the authors use RNA-seq to assess the specific GLUT isoforms expressed in the cells used in their experiments, there is a failure to investigate whether genetic Slc2a gene disruption leads to compensatory expression of other GLUT proteins. This is particularly important as the cells were sorted and expanded after lentivirus-mediated knockout of the other GLUTs prior to GLUT1 disruption. RNA-seq (or alternative method to assess for expression of each of the known GLUTs) should be done and results reported AFTER disruption of the other GLUTs.

5. Further confirmation of a lack of GLUT-mediated effects following CRISPR-mediated Slc2a disruption can be provided by assessing the effect of pharmacological GLUT inhibition, for example with cytochalasin b.

Reviewer #2: Summary: Authors utilize CRISPER-Cas 9 gene technology to ablate GLUT1 and show that 14C-glucose uptake is reduced, but that the uptake of 2-NBDG, a common fluorescent glucose that has widespread use in glucose uptake studies, is not affected. In addition, the uptake of 2-NBDG is not affected by knock out of other glucose transporters, or by ablation of select nucleoside, nucleotide, or ABC transporters. Authors conclude that 2-NBDG is taken up by cells by an unknown mechanism, but independent of glucose specificity.

Critique summary: The methodology and experimental design are appropriate, well reported and clearly described. The genetic editing technique employed in this study is a unique approach to modulate glucose transporters. The results of this study support other published work and call into question the efficacy of using 2-NBDG as a surrogate for glucose in glucose uptake studies. The data demonstrating that GLUT1 does not transport 2-NBDG is better documented than the case for the non-involvement of the other transporters studied (see below). Given the widespread use of this analog in the research literature, it is important that these results are published.

Questions and concerns:

1) I have some questions about the controls utilized in this study. The ablation of GLUT1 (Fig 1) is confirmed by both protein analysis (GLUT1 immunostaining) and by functional analysis (14C-glucose uptake). However, neither protein analysis nor functional analysis is utilized to confirm the knock out of the other putative transporters of 2-NBDG. Rather the authors rely on DNA sequencing to show the ablation of the targeted transporter and those analysis all show some unmodified targets. The actual loss of the receptor is not demonstrated. In fact, the ablation of GUT1 actually does not completely knock out GLUT1 (Fig 1), so why would we expect a complete ablation of the other transporters. I do find that data demonstrating no increase in 2NBDG negative cells convincing that the receptor was not involved in transport. (I would have expected that a certain population of cells would have both alleles ablated and thus, if that receptor were involved in transport, those cells should show up as 2NBDG negative.). Please comment on why receptor analysis and function are not reported for transporters other than GLUT1.

2) I would revise the conclusion that ‘2NBDG is actively transported’ (line 66 of introduction). ‘Active’ implies energy input required (eg ATP) for which no evidence is provided. Also, I am not entirely convinced that the data can distinguish between an actual transport process as opposed to a binding and internalization via protein recycling. The 1NBDGF control does demonstrate mediated uptake, but not the mechanism. It would have been interesting to measure uptake of just the chromophore (NBD), which I expect would be lipid soluble. The role of the fluorescent chromophore in regulating uptake is not clear.

3) Please define TPM and MFI in figure legends. Also, while stated in the figure legend, Fig 2B would be clearer if the spleen cells and bone marrow cells were designated on the figure itself (eg white print on the black photos). This is just a suggestion.

Reviewer #3: PLOS ONE

#PONE-D-21-38751 220205

Authors questioned if 2-deoxy-2[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-D-glucose (2-NBDG), the most widely used fluorescent derivative of D-glucose, is able to monitor D-glucose uptake through glucose transporters in a mouse-derived myeloma cell line 5TGM1.

Approximately 50% loss in GLUT1-positive cells by ablation of glucose transporter gene SLC2A1 (GLUT1) by CRISPR-Cas9 produced no significant difference in the mean fluorescence intensity of cells, when cells were incubated with 2-NBDG for up to an hour in a starved (i.e., D-glucose-free) condition, whereas the radioactivity significantly reduced by the ablation compared to control when14C-labeled D-glucose was applied to cells for 30 minutes.

2-NBDG, but not NBD-fructose in which the NBD moiety was attached to the C-1 position of fructose, was transported into both splenic and bone marrow plasma cells, suggesting a specific import mechanism of 2-NBDG operates in these cells in their experimental condition.

From RNA-seq analyses, authors identified SLCA1, SLC2A3, SLC2A5, SLC2A6, SLC2A8, and SLC50A1 as candidate glucose transporters in ex vivo bone marrow plasma cells and/or in 5TGM1 cells. However, authors stated that disruption of these genes in 5TGM1 cells failed to affect the 2-NBDG uptake. None of gene mutations in SLC29 nucleoside transporters, SLC35 nucleoside-sugar transporters, and ABC transporters could prevent the 2-NBDG uptake.

Authors concluded that 2-NBDG is actively transported into cells independently of known glucose transporters, and is not a faithful indicator of glucose transport. They also mentioned that 2-NBDG should not be used as a proxy for glucose uptake by mammalian cells.

General comments:

Since D-glucose is the most fundamental energy source for living things, cells have various uptake systems for D-glucose that are not only transporters but also such as channels, endocytosis, and internalization. These divergent uptake processes may operate either simultaneously or independently, temporally and/or in a spatially localized manner in the same cells depending on the condition. The point is that it should be separately discussed to test whether 2-NBDG is imported through GLUT and to evaluate whether 2-NBDG uptake in a particular cell is affected significantly by ablation of GLUT genes. Because the latter greatly depends on the relative functional contribution of GLUT in the D-glucose transport of the cell of interest. Authors may say that they used 14C D-glucose as a control. However, see comments below.

Of course, 2-NBDG is not identical to D-glucose, as also true in major D-glucose tracers 2-DG, FDG, and 3-O-methyl-D-glucose, indicating that we should be always cautious in interpretating results obtained when using these tracers. In my opinion, this study raises an issue of importance that 2-NBDG users may encounter when evaluating cellular uptake of D-glucose by 2-NBDG, especially through high affinity glucose transporters like GLUT1.

Of particular importance when evaluating the uptake kinetics of D-glucose and of its derivatives thorough glucose transporters is that we should examine the initial uptake process. For details, see Fig. 1 in Baldwin and colleagues (J. Biol. Chem. 256: 3685-3689, 1981). As illustrated, if D- and L-glucose uptake was evaluated for 30 minutes or 60 minutes, not only 14C-labeled D-glucose but also 3H-labeled L-glucose might have been taken up considerably, suggesting that non-stereoselective, possibly non-transporter-mediated uptake of glucose had took part in this system.

For an importance of evaluating the initial uptake process, see also Fig. 1 and Fig. 2 in Johnson and colleagues (J. Biol. Chem. 265: 6548-6551, 1990). The horizontal axis of Fig. 1 in this seminal paper is in seconds. Moreover, the uptake of 3H-labeled 3-O-mythyl-D-glucose was saturated at 60 seconds and the half time of the uptake was less than 15 seconds. A similar method has been applied for evaluating 2-NBDG uptake through GLUT2 and GLUT1 (see Fig. 2 in Yamada and colleagues, J. Biol. Chem. 275: 22278-22283, 2000).

If authors would like in this paper to draw such a strong conclusion about 2-NBDG including its kinetic property, they should at least analyze the initial uptake process of 2-NBDG into their cells in a quantitative manner using a standard kinetic analysis of glucose uptake as in the references cited above. Similar experiments should be conducted for radiolabeled tracers as well for comparison.

Or, authors should state the interpretation of their results more cautiously, being aware of the limitation of their experimental procedure.

Specific comments:

1) Microscopic images of 2-NBDG uptake into cells were presented only in Figure 2A in the present study. However, the image pattern shown is atypical, because the 2-NBDG signal was detected both in the cytosolic and the nuclear compartment. Indeed, authors stated, “2-NBDG was distributed evenly across the cytosol of cells”. Usually, 2-NBDG signal is mainly localized in the cytosolic compartment that could be easily discriminated from the nuclear compartment. Authors should provide a higher resolution microscopic images for showing cellular localization of 2-NBDG with the condition used in the present study. It would be possible, since authors used an imaging flow cytometer (Imagestream Mk II, Luminex) equipped with a 60x objective lens. Then, the fluorescence intensity should be evaluated for ROIs assigned to the cytosolic compartment excluding nuclei.

2) When evaluating tumor cell lines especially when they were sub-cultured for many years, multiple non-transporter-mediated uptake processes of D-glucose may operate, or dominate in some cases, in addition to glucose transporters, even if short incubation period was used. To see details in the uptake among conditions illustrated, plot the 2-NBDG intensity in Figure 1C in a linear scale, even if it will cause a change in the shape of the background intensity profile.

3) In Figure 1D, authors combined data of the 2-NBDG uptake (the mean fluorescence intensity) for an incubation periods 15, 30, 45, and 60 minutes together. Authors should separately compare the distribution of the 2-NBDG mean fluorescence intensity for 0 minutes and 15 minutes in an expanded scale to see details more clearly in a relatively short incubation period.

4) Authors used a monoclonal antibody SPM498 (Thermo Fisher Scientific) for validating the protein expression of GLUT1 on 5TGM1 cells. However, no microscopic image for the GLUT1 expression pattern was shown. This is important. Because a membrane-spanning transporter GLUT1 should be detected on the plasma membrane of cells as shown in Figure 1 of Ogorevc et al., Biomed. Rep. 2021, https://doi.org/10.3892/br.2021.1455. In this literature, the expression of GLUT1 was evaluated by the same SPM498 and compared among human tissues, while the expression on erythrocytes present in the blood vessels inside the tissues was used as a standard to determine the staining condition. As known well, immunostaining depends critically on the antibody and methods used. Thus, it is required to show that the GLUT1 immunoreactivity is detected on the plasma membrane of these 5TGM1 cells in the immunostaining condition used. Showing the expression profiles of positive and negative control cells or tissues with the same staining condition is a minimum requirement for validating that GLUT1 expression experiment was done properly. Authors should also present specimens that show how the GLUT1 immunoreactivity is affected by the SLC2A1 gene ablation in the same staining condition.

5) In Figure 4B, although the logarithmic plot of 2-NBDG uptake somewhat obscured the difference, it appears that 2-NBDG uptake in the control gRNA is larger than Slc2a1/2/3/6/8 gRNA. Plot the 2-NBDG uptake in Figure 4B in a linear scale. Similarly, in Figure 4C, the mean fluorescence intensity of 2-NBDG is larger in control than in Slc2a1/3/5/6/8. Consistently, Figure 4D shows that %2-NBDG-negative cells appears to be larger in Slc2a1/3/5/6/8 than in control. All these data may show an effect of the gene ablation on the uptake of 2-NBDG, although authors mentioned that the difference was not significant. For the statistical analyses, authors used the Mann-Whitney non-parametric t-test. First, present a scattergram that shows the distribution of actual values for Figure 4C and 4D in supplementary information. Next indicate the number of specimens tested explicitly on the bar in Figure 4C and 4D, or in the legend. Explain the rationale why authors did not use simple paired t-test in Figure 4C and 4D?

6) For evaluating the uptake of 2-NBDG, authors used incubation period longer than 15 minutes in a starved (i.e., D-glucose-free) condition at 37°C. This may activate physiological/pathophysiological processes including internalization of proteins as well as other multiple plasma membrane transporting processes. The kinetic analysis of the initial uptake within 1 minutes would provide an opportunity to identify fast uptake separately from other relatively slow processes.

7) 2-NBDG entry may occur through an opening of GAP junction/hemichannels in some neoplastic cells as well as starved normal cells (Rouach N. et. al., Science 322: 1551-1555, 2008; Thompson, RJ. et. al., Science 312: 924-927, 2008; Gandhi, GK. et. al., J. Neurochem. 110: 857-869, 2009). Authors should also test whether carbenoxolone, a widely used GAP junction/hemichannel blocker, affects the 2-NBDG entry into 5TGM1 cells in the present experimental condition.

8) Authors used 2-NBDG of Cayman Chemical (Item No. 11046). The technical information of this item No. 11046 said that the solubility of this 2-NBDG in PBS (pH 7.2) is 10 mg/ml. However, a purified 2-NBDG is reliably dissolved in aqueous solution at a concentration of approximately 1 mg/ml due to its lipophilic moiety. In our experiments, to increase the solubility, some commercially available 2-NBDG contained a solubilizing agent that potentially affect the membrane transport properties.

9) Details for the 14C D-glucose (Perkin Elmer) used in the present study should be shown, because there are different types of 14C D-glucose in this manufacturer.

Reviewer #4: The major issue with the manuscript is the sweeping claim that 2NBDG should not be used to report on glucose uptake in mammalian cells when only one cell line is used and, to boot, the one cell line used is a plasma cell line not representative of the numerous different mammalian cell types. The authors must walk back their claim about the suitability of 2NBDG to report on glucose uptake, or repeat the experiments in the manuscript on additional cell lines (both malignant and non-malignant) that are representative of all mammalian cells. Specific comments and questions are below.

The authors claim that 2NBDG is likely taken up via a transporter based on Figure 2A and a comparison to mCherry. Why does this data suggest 2NBDG is taken up by a transporter?

How was percent positive determined in Figure 2B? It looks like not all plasma cells even take up 2NBDG based on this data, which is surprising.

In Figure 3, why was C-glucose not used to confirm knockdown of the glucose transporters affected glucose uptake? There is no appropriate control shown.

There is a discrepancy in the % positive cell data shown in Figure 2D and % negative cell data in Figure 3D. How is there well below 100% positive cells in Figure 2D but nearly 0 % negative cells in 3D?

Numerous studies have shown that D-glucose competes with and reduces uptake of 2NBDG. Competition assays are needed to demonstrate that 2NBDG is actually not a reporter on glucose uptake, which is the primary claim the authors make. It is this reviewers opinion, that all that can be said from the performed study is that the authors did not find a 2NBDG transporter in plasma cells. Wording needs to be much more specific and related to the data shown rather than a sweeping claim about 2NBDG not reporting on glucose uptake.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: Manuscript review 2-NBDG.docx

Click here for additional data file.^{(16.8KB, docx)}

PLoS One. 2022 Aug 24;17(8):e0261801. doi: 10.1371/journal.pone.0261801.r002

Author response to Decision Letter 0

24 May 2022

We are grateful to the editors and reviewers for their suggestions to our manuscript. They have led us to provide a more thorough characterization of 2NBDG uptake in cells and reinforces our initial hypothesis that its transport occurs independently of glucose transporters. Changes to the manuscript have been shown in purple.

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

We apologize for the oversight and have formatted the manuscript to reflect the PLoS One’s style requirements and file naming.

2. Thank you for stating in your Funding Statement:

(This work was supported by NIH grant R01AI129945 (D.B.). L. D. was supported by a Bio5 Postdoctoral fellowship award. The use of the Imagestream was made possible by the NIH award S10 OD028466. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript)

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

We now include a statement stating the absence of any additional external funding to this study in both the manuscript and cover letter.

[Note: HTML markup is below. Please do not edit.]

We have removed instances where data has not been shown and have reworded the manuscript to reflect that.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

________________________________________

5. Review Comments to the Author

We thank this reviewer for their critique of our manuscript. Our response to their suggestions is as follows:

Specific comments and critique:

New data to address this are now shown in a new figure 9A. We quantified 2NBDG uptake in cells and found that it reaches a steady state in less than 20 seconds. The maximum intensity titrates proportionately with the concentration of 2NBDG. Saturation was not reached at any of the concentrations we tested, thereby precluding calculation of Km and Vmax. It is possible that uptake of 2NBDG is mediated by a low-affinity transporter, and we have modified the text to reflect this possibility (page 24, lines 492-501). Further, ablation of GLUT1 as shown in figure 4C has no impact on the uptake kinetics of 2NBDG.

The flow cytometric plots reflect cells that have lost both functional copies of Slc2a1, but there are additional cells that likely have lost one copy and have a partial reduction in uptake. Thus, the percentage of cells that have completely lost GLUT1 is expected to reflect a lower bound of the impact on 14C glucose uptake. We do acknowledge the simultaneous uptake and metabolism of glucose in these assays and have discussed this point in more detail in the manuscript on page 13, lines 247-252. The discrepancy between 2-deoxyglucose and 2NBDG has been addressed previously in Sinclair L et al. (1).

We accept this criticism and have added more nuance to the text to reflect these possibilities. Though we disfavor endocytosis as the primary mechanism (due to the absence of puncta in the imaging flow cytometry analysis in figure 2) and diffusion (due to the 2NBDG uptake kinetics in 5TGM1 cells and plasma cells in supplementary figure 3 and figure 3B), we acknowledge that these results indirectly support our conclusions. Page 15, lines 287-288 and page 16, lines 312-326 now discuss these findings with more context.

New RNA-seq data to address this point are shown in supplementary figure 4. Neither deletion of Slc2a1 nor other Slc2 family members lead to an increase in expression of other Slc2 family members. Further, we see no increase in expression of Slc5a1, Slc5a2, or Slc50a1 in these datasets.

New data to address this are included in figure 4A. We observe no change in 2NBDG intensity in cultures treated with cytochalasin B. We also tested more specific GLUT1 inhibitors, WZB-117 and BAY-876 (2,3), and found similar results.

We thank the reviewer for their positive comments. The disconnect between glucose and 2NBDG uptake in our assays initially took us by surprise, but now after multiple assessments by our group and others, it is clear that this analog is not a reliable surrogate for glucose uptake in mammalian cells. Our responses to their critique are below:

Questions and concerns:

We relied on DNA sequencing to identify frame-shift mutations in the various genes we assayed as there are no commercially available flow cytometric antibodies to reliably detect these mouse transporters. This is further complicated by even fewer radio-labelled compounds to specifically measure their function. Given that flow cytometry provides single cell resolution, we agree with the comment that we would have expected increased 2NBDG-negative cell frequencies had we even partially disrupted a functionally relevant transporter.

We acknowledge this criticism and have modified the text accordingly. Line 20-21 of the abstract now indicates 2NBDG uptake to be a ‘specific mechanism unlinked from glucose transport’. We examined for uptake of the chromophore 4-Chloro-7-nitrobenzofurazan (4C7NB) and have reported it in supplementary figure 3. As pointed out by the reviewer, the dynamics of 4C7NB uptake is very different from that of 2NBDG.

We apologize for the confusion and have made the necessary changes in figures 1 for MFI (page 14, line 273) and figure 5 for TPM (page 19, line 384). TPM is short for ‘transcripts per million’ and MFI means ‘Mean fluorescent intensity’. We are not sure as to why the FACS plots showing 2NBDG uptake in splenic and bone marrow plasma cells appear to have a black background. At the time of data upload, the figures have a white background and percentage positive cells indicated in the bottom right corner.

Reviewer #3: PLOS ONE

#PONE-D-21-38751 220205

General comments:

Or, authors should state the interpretation of their results more cautiously, being aware of the limitation of their experimental procedure.

We thank the reviewer for a detailed critique of our manuscript and for suggesting additional experiments that help improve our case against the use of 2NBDG as an analog of glucose in mammalian cells. Our responses to their suggestions are as follows:

Specific comments:

New data are shown to address this issue in Figure 2A. We have quantified colocalization of 2NBDG with bona fide cytoplasmic and nuclear stains. As pointed out by the reviewer, we do observe some colocalization with the nuclear stain and 2NBDG, but much less than is observed with the cytoplasm.

Data has been re-plotted into the linear scale.

New data to address this point are shown in figure 3B and supplementary figure 3. We examined the kinetics of 2NBDG uptake at earlier time points and observed that cells reach steady state in less than 20 seconds. Further, in figure 4C, deletion of GLUT1 in 5TGM1 cells by CRISPR-Cas9 does not affect 2NBDG uptake.

New data in supplementary figure 1 shows GLUT1 staining in deleted cultures. GLUT1 localizes to the cell membrane, as demonstrated by colocalization with a surface marker CD138. This is consistent with Ogorevc M et al., Biomed. Rep. 2021. In GLUT1-negative cells in the same sample, we do not observe any surface or cytosolic GLUT1, as shown by both images and quantified by similarity morphology indices.

We have added a new graph in figure 6B in the linear scale. As indicated in the figure legend, the data and statistics are pooled from three experiments. We have carried out a paired t-test for figure 6C and D as suggested. P values observed for figure 6C is 0.1298 and for 6D is 0.0843 and have been indicated on the figure.

As indicated in the materials and methods, we carried out all 2NBDG assays (unless indicated otherwise) in complete RPMI supplemented with fetal bovine serum and as such is a glucose-sufficient environment. We have now quantified 2NBDG uptake in a glucose-free environment in figure 7A and observe no impact of excess competing D-glucose in a 60 second window.

New data to assess the role of carbenoxolone on 2NBDG uptake are shown in figure 4B. Carbenoxolone does not impact 2NBDG uptake.

As pointed out by the reviewer, 2NBDG from Cayman chemical is sold as a crystalline solid. We resuspend this compound to an initial concentration of 10mg/mL in 1x PBS and further aliquot it to 1mg/mL in 1x PBS. We observe complete solubility of the compound at both concentrations and do not add any solubilizing agent to any of the preparations. We have provided this updated description to the materials and methods section on page 6, lines 105-108.

9) Details for the 14C D-glucose (Perkin Elmer) used in the present study should be shown, because there are different types of 14C D-glucose in this manufacturer.

NEC042X as manufactured by Perkin Elmer is a uniformly 14C labelled glucose compound that is sold in three volumes. Each have the same catalog number but with an addendum to the existing alphanumeric code to reflect the volume. We have updated the materials and methods section to reflect our purchase of NEC042X050UC, the 50μCi preparation and added its specific activity, which is 275 mCi/mmol. Page 10, line 187 details this information.

We have added new data on primary plasma cells, B cells, and total spleen cells demonstrating that large excesses of competing glucose fail to inhibit 2NBDG uptake in figure 7. While we are uncertain what the reviewer means by ‘representative of all mammalian cells’, we refer to three other manuscripts that have also observed a discrepancy between 2NBDG and glucose uptake. First, the observations of Hamilton K et al., who have examined 2NBDG uptake in the mouse fibroblast cell line L929 and second by Sinclair L et al. who have found similar discrepancies in mouse primary T cells, and Reinfeld B et al., who have found discrepancies between FDG and 2NBDG uptake in vivo (1,4,5). We discuss these manuscripts in the discussion, and they serve as orthogonal confirmations of the experiments we have carried out in our manuscript.

The authors claim that 2NBDG is likely taken up via a transporter based on Figure 2A and a comparison to mCherry. Why does this data suggest 2NBDG is taken up by a transporter?

As a hydrophilic compound, uptake of 2NBDG is presumably mediated either by a transporter which delivers the compound to the cytoplasm or via endocytosis, in which the compound would be expected to localize preferentially to vesicles. Our data would indicate that there is mediated uptake, both by colocalization data in figure 2 and the presence of 2NBDG negative plasma cells in 2NBDG-injected mice in figure 3A. We have edited the text to reflect these observations on page 15, lines 279-290, page 16 lines 306-323 and page 17, lines 324-326.

How was percent positive determined in Figure 2B? It looks like not all plasma cells even take up 2NBDG based on this data, which is surprising.

We note two contours in the upper left panel of figure 3A indicative of two populations that differ in their intensity of 2NBDG (x-axis). We gate on the population with higher 2NBDG intensity as 2NBDG-positive cells and confirm this by using the same gate on a different sample of plasma cells obtained from mice not injected with 2NBDG. We refer the reviewer to our previous findings demonstrating that long lived plasma cells take up larger amounts of 2NBDG in vivo than do short-lived cells (6,7).

In Figure 3, why was C-glucose not used to confirm knockdown of the glucose transporters affected glucose uptake? There is no appropriate control shown.

To be clear, these experiments are not knockdown/shRNA assays. They are CRISPR-mediated indel mutations that create early frameshifts, validated by multiple guide RNAs targeting different regions in the gene. Null mutations can thus be easily quantified using next generation sequencing. We observed in figure 1B that ablation of GLUT1 in 5TGM1 cells induced approximately 80% reduction in 14C-glucose uptake. As shown in what is now figure 5A, most of the other Slc2 family members are expressed at quite low levels in 5TGM1 cells and are thought to preferentially mediate uptake of other sugars. Detecting further loss of glucose uptake beyond what is already seen in the absence of GLUT1 would be very challenging. We do demonstrate a high frequency of frameshift mutations in the genomic sequences of the assayed glucose transporters figure 5B. Despite this we see no impact on 2NBDG uptake.

In what is now figure 3A, we report the frequencies of percent 2NBDG positive cells in primary plasma cells from mouse spleens and bone marrows. In what is now figure 5D, we are examining frequencies of 2NBDG negative 5TGM1 cells that have been deleted for the various Slc2 family members and Slc50a1. As mentioned above, this distribution is expected for primary plasma cells.

New data indicating the influence of competing glucose on 2NBDG uptake is shown in now figure 7A. We do not observe any change in the kinetics of 2NBDG uptake in the presence of titrating amounts of glucose. While not statistically significant, if anything, 30mM and 10mM glucose in the assay medium showed higher intensity earlier than control cells in glucose-free media. We acknowledge that we were unable to find the putative transporter for 2NBDG through our experiments. However, we do provide ample evidence now that 2NBDG uptake is distinct from glucose uptake and should be treated as such. Had earlier studies provided this level of rigor, we very much doubt that this reagent would be in as widespread use as a surrogate for glucose uptake.

References

1. Sinclair LV, Barthelemy C, Cantrell DA. Single Cell Glucose Uptake Assays: A Cautionary Tale. Immunometabolism. 2020 Aug 17;2(4):e200029.

2. Siebeneicher H, Cleve A, Rehwinkel H, Neuhaus R, Heisler I, Müller T, et al. Identification and Optimization of the First Highly Selective GLUT1 Inhibitor BAY-876. ChemMedChem. 2016;11(20):2261–71.

3. Ojelabi OA, Lloyd KP, Simon AH, De Zutter JK, Carruthers A. WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site*. Journal of Biological Chemistry. 2016 Dec 1;291(52):26762–72.

4. Hamilton KE, Bouwer MF, Louters LL, Looyenga BD. Cellular binding and uptake of fluorescent glucose analogs 2-NBDG and 6-NBDG occurs independent of membrane glucose transporters. Biochimie. 2021 Nov 1;190:1–11.

5. Reinfeld BI, Madden MZ, Wolf MM, Chytil A, Bader JE, Patterson AR, et al. Cell-programmed nutrient partitioning in the tumour microenvironment. Nature. 2021 May;593(7858):282–8.

6. Lam WY, Becker AM, Kennerly KM, Wong R, Curtis JD, Llufrio EM, et al. Mitochondrial Pyruvate Import Promotes Long-Term Survival of Antibody-Secreting Plasma Cells. Immunity. 2016 Jul 19;45(1):60–73.

7. Lam WY, Jash A, Yao CH, D’Souza L, Wong R, Nunley RM, et al. Metabolic and Transcriptional Modules Independently Diversify Plasma Cell Lifespan and Function. Cell Rep. 2018 Aug 28;24(9):2479-2492.e6.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(43.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0261801.r003

Decision Letter 1

Hodaka Fujii

1 Jul 2022

PONE-D-21-38751R1Genetic evidence that uptake of the fluorescent analog 2NBDG occurs independently of known glucose transportersPLOS ONE

Dear Dr. Bhattacharya,

Please submit your revised manuscript by Aug 15 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Hodaka Fujii, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #2: (No Response)

Reviewer #3: Authors have made a considerable revision to the original manuscript. My concern is that they generalized results obtained from a limited types of cells expressing GLUT1 as "Thus, cellular uptake of 2NBDG is not a faithful indicator of glucose transport ...” (the last sentence in Abstract). I would strongly recommend authors to confine their statement to glucose transport at least through GLUT1, since no experiment was done for cells intrinsically expressing such as GLUT2.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

**********

PLoS One. 2022 Aug 24;17(8):e0261801. doi: 10.1371/journal.pone.0261801.r004

Author response to Decision Letter 1

1 Jul 2022

Journal Requirements:

We have examined all the references and found them to be complete and correct. All cited manuscripts are currently accepted and available on the publisher’s website or on PubMed Central.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: (No Response)

Reviewer #3: Yes

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: (No Response)

Reviewer #3: Yes

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: (No Response)

Reviewer #3: Yes

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: (No Response)

Reviewer #3: Yes

________________________________________

6. Review Comments to the Author

Reviewer #2: (No Response)

We apologize for our obstinacy, but even for GLUT1, we cannot conclude with certainty that 2NBDG cannot be transported through it at all. Instead, the data show that there are other glucose transporter-independent pathways by which 2NBDG predominantly enters cells. This background is what makes 2NBDG unreliable as a surrogate for glucose uptake. Whether GLUT2 or other glucose transporters are expressed does not change this fact. In this sense, we feel that it would be an overreach to adopt this suggestion and claim 2NBDG cannot be transported by GLUT1.

________________________________________

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

Attachment

Submitted filename: Response to reviewers-v5_db.docx

Click here for additional data file.^{(16.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0261801.r005

Decision Letter 2

Hodaka Fujii

1 Aug 2022

PONE-D-21-38751R2Genetic evidence that uptake of the fluorescent analog 2NBDG occurs independently of known glucose transportersPLOS ONE

Dear Dr. Bhattacharya,

One of the reviewers still raised some concerns about the wordings of your revised manuscript. In this regard, I don't see any problem in the wordings of the title because your data showed that there exist some mechanisms of uptake of 2NBDG independent of known glucose transporters. On the other hand, the last sentences of the Abstract and Discussion might be very strong statements. Can you consider making their expression a little milder? For example, the last sentence of the Abstract can be "Thus, cellular uptake of 2NBDG is not necessarily a faithful indicator of glucose transport...", and the last sentence of the Discussion can be "...., we advise that despite their convenience, it should not be considered as direct indicator of glucose uptake." I think that researchers can use the 2NBDG uptake assay but should carefully interpret their results.

Please submit your revised manuscript by Sep 15 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Hodaka Fujii, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #3: This reviewer agrees an involvement of non-GLUT1-mediated mechanism in the uptake of 2-NBDG into certain cell types. In this respect, this manuscript provides valuable information to the scientific community. However, this reviewer unfortunately can not accept authors' conclusion and the title in the present form. Authors' evidence was obtained from limited cell types and was limited to GLUT1. However, the title of the manuscript included sentence "uptake of the fluorescent analog 2NBDG occurs independently of 'known' glucose transporters". This title would not be adequate, if 2-NBDG uptake occurs through GLUT2 in other cell lines to a significant extent. To greatly increase values of this important manuscript, this reviewer recommends authors to refine the title and the conclusive sentence, or at least to mention GLUT2.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

**********

PLoS One. 2022 Aug 24;17(8):e0261801. doi: 10.1371/journal.pone.0261801.r006

Author response to Decision Letter 2

2 Aug 2022

As suggested by the editor, we have modified the abstract to now end with the statement, “Thus, cellular uptake of 2NBDG is not necessarily a faithful indicator of glucose transport and is promoted by an unknown mechanism.” Further, the discussion now concludes the manuscript with the statement, “… we strongly advise that despite their convenience, these assays should not be considered as direct indicators of glucose uptake.”

Attachment

Submitted filename: Response to reviewers-v5.docx

Click here for additional data file.^{(15.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0261801.r007

Decision Letter 3

Hodaka Fujii

11 Aug 2022

Genetic evidence that uptake of the fluorescent analog 2NBDG occurs independently of known glucose transporters

PONE-D-21-38751R3

Dear Dr. Bhattacharya,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Hodaka Fujii, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0261801.r008

Acceptance letter

Hodaka Fujii

15 Aug 2022

PONE-D-21-38751R3

Genetic evidence that uptake of the fluorescent analog 2NBDG occurs independently of known glucose transporters

Dear Dr. Bhattacharya:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Hodaka Fujii

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Slc2a1 deletion leads to a loss of surface GLUT1 expression.

(TIF)

Click here for additional data file.^{(523.9KB, tif)}

S2 Fig. Structures of 2NBDG and 1NBDF in relation to glucose and naturally occurring nucleosides.

Chemical structures of (A) D-Glucose, (B) 2NBDG, (C) 1NBDF, (D) 4C7NB, (E) Guanosine, (F) Adenosine, (G) Deoxythymidine, (H) Uridine, and (I) Cytosine. Structures generated using ChemDraw v.20.1.1.

(TIF)

Click here for additional data file.^{(347.1KB, tif)}

(TIF)

Click here for additional data file.^{(206.5KB, tif)}

S4 Fig. Slc2 deletion does not lead to compensatory expression of other transporters.

(TIF)

Click here for additional data file.^{(310.3KB, tif)}

Attachment

Submitted filename: Manuscript review 2-NBDG.docx

Click here for additional data file.^{(16.8KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(43.8KB, docx)}

Attachment

Submitted filename: Response to reviewers-v5_db.docx

Click here for additional data file.^{(16.5KB, docx)}

Attachment

Submitted filename: Response to reviewers-v5.docx

Click here for additional data file.^{(15.1KB, docx)}

Data Availability Statement

RNA-seq data reported in this paper is available at NCBI GEO (accession GSE202181). All other relevant data are within the manuscript and its Supporting Information files.

[pone.0261801.ref001] 1.Nelson DL, Cox MM. Lehninger Principles of Biochemistry. 7th ed. New York: W. H. Freeman; 2017. [Google Scholar]

[pone.0261801.ref002] 2.Lam WY, Bhattacharya D. Metabolic Links between Plasma Cell Survival, Secretion, and Stress. Trends in Immunology. 2018;39: 19–27. doi: 10.1016/j.it.2017.08.007 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref003] 3.Som P, Atkins HL, Bandoypadhyay D, Fowler JS, MacGregor RR, Matsui K, et al. A Fluorinated Glucose Analog, 2-fluoro-2-deoxy-D-glucose (F-18): Nontoxic Tracer for Rapid Tumor Detection. Journal of Nuclear Medicine. 1980;21: 670–675. [PubMed] [Google Scholar]

[pone.0261801.ref004] 4.Yamamoto N, Ueda-Wakagi M, Sato T, Kawasaki K, Sawada K, Kawabata K, et al. Measurement of Glucose Uptake in Cultured Cells. Current Protocols in Pharmacology. 2015;71: 12.14.1–12.14.26. doi: 10.1002/0471141755.ph1214s71 [DOI] [PubMed] [Google Scholar]

[pone.0261801.ref005] 5.Maric T, Mikhaylov G, Khodakivskyi P, Bazhin A, Sinisi R, Bonhoure N, et al. Bioluminescent-based imaging and quantification of glucose uptake in vivo. Nat Methods. 2019;16: 526–532. doi: 10.1038/s41592-019-0421-z [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref006] 6.Kim WH, Lee J, Jung D-W, Williams DR. Visualizing Sweetness: Increasingly Diverse Applications for Fluorescent-Tagged Glucose Bioprobes and Their Recent Structural Modifications. Sensors. 2012;12: 5005–5027. doi: 10.3390/s120405005 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref007] 7.Yoshioka K, Takahashi H, Homma T, Saito M, Oh KB, Nemoto Y, et al. A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli. Biochim Biophys Acta. 1996;1289: 5–9. doi: 10.1016/0304-4165(95)00153-0 [DOI] [PubMed] [Google Scholar]

[pone.0261801.ref008] 8.Tao J, Diaz RK, Teixeira CRV, Hackmann TJ. Transport of a Fluorescent Analogue of Glucose (2-NBDG) versus Radiolabeled Sugars by Rumen Bacteria and Escherichia coli. Biochemistry. 2016;55: 2578–2589. doi: 10.1021/acs.biochem.5b01286 [DOI] [PubMed] [Google Scholar]

[pone.0261801.ref009] 9.Lam WY, Jash A, Yao C-H, D’Souza L, Wong R, Nunley RM, et al. Metabolic and Transcriptional Modules Independently Diversify Plasma Cell Lifespan and Function. Cell Rep. 2018;24: 2479–2492.e6. doi: 10.1016/j.celrep.2018.07.084 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref010] 10.Lam WY, Becker AM, Kennerly KM, Wong R, Curtis JD, Llufrio EM, et al. Mitochondrial Pyruvate Import Promotes Long-Term Survival of Antibody-Secreting Plasma Cells. Immunity. 2016;45: 60–73. doi: 10.1016/j.immuni.2016.06.011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref011] 11.D’Souza L, Bhattacharya D. Plasma cells: You are what you eat. Immunological Reviews. 2019;288: 161–177. doi: 10.1111/imr.12732 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref012] 12.Deng D, Yan N. GLUT, SGLT, and SWEET: Structural and mechanistic investigations of the glucose transporters. Protein Science. 2016;25: 546–558. doi: 10.1002/pro.2858 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref013] 13.Chen L-Q, Hou B-H, Lalonde S, Takanaga H, Hartung ML, Qu X-Q, et al. Sugar transporters for intercellular exchange and nutrition of pathogens. Nature. 2010;468: 527–532. doi: 10.1038/nature09606 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref014] 14.Caro-Maldonado A, Wang R, Nichols AG, Kuraoka M, Milasta S, Sun LD, et al. Metabolic reprogramming is required for antibody production that is suppressed in anergic but exaggerated in chronically BAFF-exposed B cells. J Immunol. 2014;192: 3626–3636. doi: 10.4049/jimmunol.1302062 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref015] 15.Sinclair LV, Barthelemy C, Cantrell DA. Single Cell Glucose Uptake Assays: A Cautionary Tale. Immunometabolism. 2020;2: e200029. doi: 10.20900/immunometab20200029 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref016] 16.Hamilton KE, Bouwer MF, Louters LL, Looyenga BD. Cellular binding and uptake of fluorescent glucose analogs 2-NBDG and 6-NBDG occurs independent of membrane glucose transporters. Biochimie. 2021;190: 1–11. doi: 10.1016/j.biochi.2021.06.017 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref017] 17.Reinfeld BI, Madden MZ, Wolf MM, Chytil A, Bader JE, Patterson AR, et al. Cell-programmed nutrient partitioning in the tumour microenvironment. Nature. 2021;593: 282–288. doi: 10.1038/s41586-021-03442-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref018] 18.Garrett IR, Dallas S, Radl J, Mundy GR. A murine model of human myeloma bone disease. Bone. 1997;20: 515–520. doi: 10.1016/s8756-3282(97)00056-2 [DOI] [PubMed] [Google Scholar]

[pone.0261801.ref019] 19.Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-Guided Human Genome Engineering via Cas9. Science. 2013;339: 823–826. doi: 10.1126/science.1232033 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref020] 20.Sanson KR, Hanna RE, Hegde M, Donovan KF, Strand C, Sullender ME, et al. Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun. 2018;9: 5416. doi: 10.1038/s41467-018-07901-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref021] 21.Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34: 184–191. doi: 10.1038/nbt.3437 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref022] 22.Clement K, Rees H, Canver MC, Gehrke JM, Farouni R, Hsu JY, et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol. 2019;37: 224–226. doi: 10.1038/s41587-019-0032-3 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref023] 23.Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017;14: 417–419. doi: 10.1038/nmeth.4197 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref024] 24.Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15: 550. doi: 10.1186/s13059-014-0550-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref025] 25.Afgan E, Baker D, van den Beek M, Blankenberg D, Bouvier D, Čech M, et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Research. 2016;44: W3–W10. doi: 10.1093/nar/gkw343 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref026] 26.Wardell SE, Ilkayeva OR, Wieman HL, Frigo DE, Rathmell JC, Newgard CB, et al. Glucose Metabolism as a Target of Histone Deacetylase Inhibitors. Molecular Endocrinology. 2009;23: 388–401. doi: 10.1210/me.2008-0179 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref027] 27.Ogorevc M, Strikic A, Tomas SZ. Determining the immunohistochemical expression of GLUT1 in renal cell carcinoma using the HSCORE method. Biomed Rep. 2021;15: 79. doi: 10.3892/br.2021.1455 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref028] 28.Levi J, Cheng Z, Gheysens O, Patel M, Chan CT, Wang Y, et al. Fluorescent fructose derivatives for imaging breast cancer cells. Bioconjug Chem. 2007;18: 628–634. doi: 10.1021/bc060184s [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref029] 29.Devés R, Krupka RM. Cytochalasin B and the kinetics of inhibition of biological transport. A case of asymmetric binding to the glucose carrier. Biochimica et Biophysica Acta (BBA)—Biomembranes. 1978;510: 339–348. doi: 10.1016/0005-2736(78)90034-2 [DOI] [PubMed] [Google Scholar]

[pone.0261801.ref030] 30.Mizel SB, Wilson L. Inhibition of the Transport of Several Hexoses in Mammalian Cells by Cytochalasin B. Journal of Biological Chemistry. 1972;247: 4102–4105. doi: 10.1016/S0021-9258(19)45146-6 [DOI] [PubMed] [Google Scholar]

[pone.0261801.ref031] 31.MacLean-Fletcher S, Pollard TD. Mechanism of action of cytochalasin B on actin. Cell. 1980;20: 329–341. doi: 10.1016/0092-8674(80)90619-4 [DOI] [PubMed] [Google Scholar]

[pone.0261801.ref032] 32.Siebeneicher H, Cleve A, Rehwinkel H, Neuhaus R, Heisler I, Müller T, et al. Identification and Optimization of the First Highly Selective GLUT1 Inhibitor BAY-876. ChemMedChem. 2016;11: 2261–2271. doi: 10.1002/cmdc.201600276 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref033] 33.Ojelabi OA, Lloyd KP, Simon AH, De Zutter JK, Carruthers A. WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site*. Journal of Biological Chemistry. 2016;291: 26762–26772. doi: 10.1074/jbc.M116.759175 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref034] 34.Davidson JS, Baumgarten IM, Harley EH. Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid. Biochemical and Biophysical Research Communications. 1986;134: 29–36. doi: 10.1016/0006-291x(86)90522-x [DOI] [PubMed] [Google Scholar]

[pone.0261801.ref035] 35.Rouach N, Koulakoff A, Abudara V, Willecke K, Giaume C. Astroglial Metabolic Networks Sustain Hippocampal Synaptic Transmission. Science. 2008;322: 1551–1555. doi: 10.1126/science.1164022 [DOI] [PubMed] [Google Scholar]

[pone.0261801.ref036] 36.Trotter TN, Li M, Pan Q, Peker D, Rowan PD, Li J, et al. Myeloma cell-derived Runx2 promotes myeloma progression in bone. Blood. 2015;125: 3598–3608. doi: 10.1182/blood-2014-12-613968 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0261801.ref037] 37.Wright EM, Turk E. The sodium/glucose cotransport family SLC5. Pflugers Arch—Eur J Physiol. 2004;447: 510–518. doi: 10.1007/s00424-003-1063-6 [DOI] [PubMed] [Google Scholar]

[pone.0261801.ref038] 38.Liong E, Kong SK, Au KK, Li JY, Xu GY, Lee YL, et al. Inhibition of glucose uptake and suppression of glucose transporter 1 mRNA expression in L929 cells by tumour necrosis factor-α. Life Sciences. 1999;65: PL215–PL220. doi: 10.1016/S0024-3205(99)00408-7 [DOI] [PubMed] [Google Scholar]

PERMALINK

Genetic evidence that uptake of the fluorescent analog 2NBDG occurs independently of known glucose transporters

Lucas J D’Souza

Stephen H Wright

Deepta Bhattacharya

Roles

Abstract

Introduction

Materials and methods

Ethics statement

Mice

Primary cells and cell lines

Chemicals and cell culture media

Plasmids and lentivirus generation

Flow cytometry

Imaging flow cytometry

14C-Glucose uptake assays

Next generation sequencing

RNA sequencing

Statistical analysis

Results

GLUT1 does not mediate uptake of 2NBDG

Fig 1. 2NBDG uptake is unaffected by Slc2a1 deletion in 5TGM1 cells.

Plasma cells take up 2NBDG, but not 1-NBD-Fructose

Fig 2. Plasma cells take up 2NBDG and retain it in the cytosol.

Fig 3. Plasma cells specifically transport 2NBDG.

GLUT1 inhibition or ablation does not affect 2NBDG uptake kinetics

Fig 4. The kinetics of 2NBDG uptake is unaffected by GLUT1 inhibition or deletion.

2NBDG uptake does not depend on other glucose transporters

Fig 5. 2NBDG uptake takes place independently of sugar transporters.

Fig 6. Combined deletion of sugar transporters does not affect 2NBDG uptake.

Exogenous glucose does not inhibit 2NBDG uptake

Fig 7. 2NBDG uptake in cells is independent of competing glucose.

Nucleoside and nucleoside-sugar transporters do not transport 2NBDG

Fig 8. 2NBDG is not imported through nucleoside or nucleoside-sugar transporters.

2NBDG uptake is a low-affinity, sodium independent process

Fig 9. 2NBDG transport is a low affinity process.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Hodaka Fujii

Roles

Author response to Decision Letter 0

Decision Letter 1

Hodaka Fujii

Roles

Author response to Decision Letter 1

Decision Letter 2

Hodaka Fujii

Roles

Author response to Decision Letter 2

Decision Letter 3

Hodaka Fujii

Roles

Acceptance letter

Hodaka Fujii

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

¹⁴C-Glucose uptake assays