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Characterization of a fluorescent glucose derivative 1-NBDG and its application in the identification of natural SGLT1/2 inhibitors

Glucose is an important energy source for cells. Glucose transport is mediated by two types of glucose transporters: the active sodium-coupled glucose cotransporters (SGLTs), and the passive glucose transporters (GLUTs). Development of an easy way to detect glucose uptake by the cell can be valuable...

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Autores principales: Kao, Ting-Yu, Wu, Hwa-Wei, Lee, Shoei-Sheng, Liang, Pi-Hui, Guh, Jih-Hwa, Hsu, Lih-Ching
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taiwan Food and Drug Administration 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9261792/
https://www.ncbi.nlm.nih.gov/pubmed/35696243
http://dx.doi.org/10.38212/2224-6614.3371
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author Kao, Ting-Yu
Wu, Hwa-Wei
Lee, Shoei-Sheng
Liang, Pi-Hui
Guh, Jih-Hwa
Hsu, Lih-Ching
author_facet Kao, Ting-Yu
Wu, Hwa-Wei
Lee, Shoei-Sheng
Liang, Pi-Hui
Guh, Jih-Hwa
Hsu, Lih-Ching
author_sort Kao, Ting-Yu
collection PubMed
description Glucose is an important energy source for cells. Glucose transport is mediated by two types of glucose transporters: the active sodium-coupled glucose cotransporters (SGLTs), and the passive glucose transporters (GLUTs). Development of an easy way to detect glucose uptake by the cell can be valuable for research. 1-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-1-deoxy-d-glucose (1-NBDG) is a newly synthesized fluorescent glucose analogue. Unlike 2-NBDG, which is a good substrate of GLUTs but not SGLTs, 1-NBDG can be transported by both GLUTs and SGLTs. Thus, 1-NBDG is useful for the screening of SGLT1 and SGLT2 inhibitors. Here we further characterized 1-NBDG and compared it with 2-NBDG. The fluorescence of both 1-NBDG and 2-NBDG was quenched under alkaline conditions, but only 1-NBDG fluorescence could be restored upon neutralization. HPLC analysis revealed that 2-NBDG was decomposed leading to loss of fluorescence, whereas 1-NBDG remained intact in a NaOH solution. Thus, after cellular uptake, 1-NBDG fluorescence can be detected on a plate reader simply by cell lysis in a NaOH solution followed by neutralization with an HCl solution. The fluorescence stability of 1-NBDG was stable for up to 5 h once cells were lysed; however, similar to 2-NBDG, intracellular 1-NBDG was not stable and the fluorescence diminished substantially within one hour. 1-NBDG uptake could also be detected at the single cell level and inhibition of 1-NBDG uptake by SGLT inhibitors could be detected by flow cytometry. Furthermore, 1-NBDG was successfully used in a high-throughput cell-based method to screen for potential SGLT1 and SGLT2 inhibitors. The SGLT inhibitory activities of 67 flavonoids and flavonoid glycosides purified from plants were evaluated and several selective SGLT1, selective SGLT2, as well as dual SGLT1/2 inhibitors were identified. Structure-activity relationship analysis revealed that glycosyl residues were crucial since the aglycon showed no SGLT inhibitory activities. In addition, the sugar inter-linkage and their substitution positions to the aglycon affected not only the inhibitory activities but also the selectivity toward SGLT1 and SGLT2.
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spelling pubmed-92617922022-07-18 Characterization of a fluorescent glucose derivative 1-NBDG and its application in the identification of natural SGLT1/2 inhibitors Kao, Ting-Yu Wu, Hwa-Wei Lee, Shoei-Sheng Liang, Pi-Hui Guh, Jih-Hwa Hsu, Lih-Ching J Food Drug Anal Original Article Glucose is an important energy source for cells. Glucose transport is mediated by two types of glucose transporters: the active sodium-coupled glucose cotransporters (SGLTs), and the passive glucose transporters (GLUTs). Development of an easy way to detect glucose uptake by the cell can be valuable for research. 1-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-1-deoxy-d-glucose (1-NBDG) is a newly synthesized fluorescent glucose analogue. Unlike 2-NBDG, which is a good substrate of GLUTs but not SGLTs, 1-NBDG can be transported by both GLUTs and SGLTs. Thus, 1-NBDG is useful for the screening of SGLT1 and SGLT2 inhibitors. Here we further characterized 1-NBDG and compared it with 2-NBDG. The fluorescence of both 1-NBDG and 2-NBDG was quenched under alkaline conditions, but only 1-NBDG fluorescence could be restored upon neutralization. HPLC analysis revealed that 2-NBDG was decomposed leading to loss of fluorescence, whereas 1-NBDG remained intact in a NaOH solution. Thus, after cellular uptake, 1-NBDG fluorescence can be detected on a plate reader simply by cell lysis in a NaOH solution followed by neutralization with an HCl solution. The fluorescence stability of 1-NBDG was stable for up to 5 h once cells were lysed; however, similar to 2-NBDG, intracellular 1-NBDG was not stable and the fluorescence diminished substantially within one hour. 1-NBDG uptake could also be detected at the single cell level and inhibition of 1-NBDG uptake by SGLT inhibitors could be detected by flow cytometry. Furthermore, 1-NBDG was successfully used in a high-throughput cell-based method to screen for potential SGLT1 and SGLT2 inhibitors. The SGLT inhibitory activities of 67 flavonoids and flavonoid glycosides purified from plants were evaluated and several selective SGLT1, selective SGLT2, as well as dual SGLT1/2 inhibitors were identified. Structure-activity relationship analysis revealed that glycosyl residues were crucial since the aglycon showed no SGLT inhibitory activities. In addition, the sugar inter-linkage and their substitution positions to the aglycon affected not only the inhibitory activities but also the selectivity toward SGLT1 and SGLT2. Taiwan Food and Drug Administration 2021-09-15 /pmc/articles/PMC9261792/ /pubmed/35696243 http://dx.doi.org/10.38212/2224-6614.3371 Text en © 2021 Taiwan Food and Drug Administration https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC-BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) ).
spellingShingle Original Article
Kao, Ting-Yu
Wu, Hwa-Wei
Lee, Shoei-Sheng
Liang, Pi-Hui
Guh, Jih-Hwa
Hsu, Lih-Ching
Characterization of a fluorescent glucose derivative 1-NBDG and its application in the identification of natural SGLT1/2 inhibitors
title Characterization of a fluorescent glucose derivative 1-NBDG and its application in the identification of natural SGLT1/2 inhibitors
title_full Characterization of a fluorescent glucose derivative 1-NBDG and its application in the identification of natural SGLT1/2 inhibitors
title_fullStr Characterization of a fluorescent glucose derivative 1-NBDG and its application in the identification of natural SGLT1/2 inhibitors
title_full_unstemmed Characterization of a fluorescent glucose derivative 1-NBDG and its application in the identification of natural SGLT1/2 inhibitors
title_short Characterization of a fluorescent glucose derivative 1-NBDG and its application in the identification of natural SGLT1/2 inhibitors
title_sort characterization of a fluorescent glucose derivative 1-nbdg and its application in the identification of natural sglt1/2 inhibitors
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9261792/
https://www.ncbi.nlm.nih.gov/pubmed/35696243
http://dx.doi.org/10.38212/2224-6614.3371
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