Incorporation of Fluorescence Ceramide-Based HPLC Assay for Rapidly and Efficiently Assessing Glucosylceramide Synthase In Vivo

Abstract Glucosylceramide synthase (GCS) is a rate-limiting enzyme catalyzing ceramide glycosylation, thereby regulating cellular ceramide levels and the synthesis of glycosphingolipids (GSLs) in cellular membranes. Alterations of GCS not only affect membrane integrity, but also closely correlate wi...

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Autores principales:	Sachin K. Khiste, Salman B. Hosain, Yixuan Dong, Mohammad B. Uddin, Kartik R. Roy, Ronald A. Hill, Zhijun Liu, Yong-Yu Liu
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/524f0c7128c14f34911c53c9faa60238
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spelling	oai:doaj.org-article:524f0c7128c14f34911c53c9faa602382021-12-02T12:32:45ZIncorporation of Fluorescence Ceramide-Based HPLC Assay for Rapidly and Efficiently Assessing Glucosylceramide Synthase In Vivo10.1038/s41598-017-03320-92045-2322https://doaj.org/article/524f0c7128c14f34911c53c9faa602382017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03320-9https://doaj.org/toc/2045-2322Abstract Glucosylceramide synthase (GCS) is a rate-limiting enzyme catalyzing ceramide glycosylation, thereby regulating cellular ceramide levels and the synthesis of glycosphingolipids (GSLs) in cellular membranes. Alterations of GCS not only affect membrane integrity, but also closely correlate with stem cell pluripotency, cancer drug resistance, GSL storage disorders and other diseases. Enzyme activities measured conventionally with currently available ex-vivo methods do not enable reliable assessment of the roles played by GCS in vivo. We report herein a substrate-incorporation method enabling rapid and efficient assessment of GCS in-vivo activity. Upon nanoparticle-based delivery, fluorescent NBD C6-ceramide was efficiently converted to NBD C6-glucosylceramide in live cells or in mouse tissues, whereupon an HPLC assay enabled detection and quantification of NBD C6-glucosylceramide in the low-femtomolar range. The enzyme kinetics of GCS in live cells and mouse liver were well-described by the Michaelis-Menten model. GCS activities were significantly higher in drug-resistant cancer cells and in tumors overexpressing GCS, but reduced after silencing GCS expression or inhibiting this enzyme. Our studies indicate that this rapid and efficient method provides a valuable means for accurately assessing the roles played by GCS in normal vs. pathological states, including ones involving cancer drug resistance.Sachin K. KhisteSalman B. HosainYixuan DongMohammad B. UddinKartik R. RoyRonald A. HillZhijun LiuYong-Yu LiuNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-13 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Sachin K. Khiste Salman B. Hosain Yixuan Dong Mohammad B. Uddin Kartik R. Roy Ronald A. Hill Zhijun Liu Yong-Yu Liu Incorporation of Fluorescence Ceramide-Based HPLC Assay for Rapidly and Efficiently Assessing Glucosylceramide Synthase In Vivo
description	Abstract Glucosylceramide synthase (GCS) is a rate-limiting enzyme catalyzing ceramide glycosylation, thereby regulating cellular ceramide levels and the synthesis of glycosphingolipids (GSLs) in cellular membranes. Alterations of GCS not only affect membrane integrity, but also closely correlate with stem cell pluripotency, cancer drug resistance, GSL storage disorders and other diseases. Enzyme activities measured conventionally with currently available ex-vivo methods do not enable reliable assessment of the roles played by GCS in vivo. We report herein a substrate-incorporation method enabling rapid and efficient assessment of GCS in-vivo activity. Upon nanoparticle-based delivery, fluorescent NBD C6-ceramide was efficiently converted to NBD C6-glucosylceramide in live cells or in mouse tissues, whereupon an HPLC assay enabled detection and quantification of NBD C6-glucosylceramide in the low-femtomolar range. The enzyme kinetics of GCS in live cells and mouse liver were well-described by the Michaelis-Menten model. GCS activities were significantly higher in drug-resistant cancer cells and in tumors overexpressing GCS, but reduced after silencing GCS expression or inhibiting this enzyme. Our studies indicate that this rapid and efficient method provides a valuable means for accurately assessing the roles played by GCS in normal vs. pathological states, including ones involving cancer drug resistance.
format	article
author	Sachin K. Khiste Salman B. Hosain Yixuan Dong Mohammad B. Uddin Kartik R. Roy Ronald A. Hill Zhijun Liu Yong-Yu Liu
author_facet	Sachin K. Khiste Salman B. Hosain Yixuan Dong Mohammad B. Uddin Kartik R. Roy Ronald A. Hill Zhijun Liu Yong-Yu Liu
author_sort	Sachin K. Khiste
title	Incorporation of Fluorescence Ceramide-Based HPLC Assay for Rapidly and Efficiently Assessing Glucosylceramide Synthase In Vivo
title_short	Incorporation of Fluorescence Ceramide-Based HPLC Assay for Rapidly and Efficiently Assessing Glucosylceramide Synthase In Vivo
title_full	Incorporation of Fluorescence Ceramide-Based HPLC Assay for Rapidly and Efficiently Assessing Glucosylceramide Synthase In Vivo
title_fullStr	Incorporation of Fluorescence Ceramide-Based HPLC Assay for Rapidly and Efficiently Assessing Glucosylceramide Synthase In Vivo
title_full_unstemmed	Incorporation of Fluorescence Ceramide-Based HPLC Assay for Rapidly and Efficiently Assessing Glucosylceramide Synthase In Vivo
title_sort	incorporation of fluorescence ceramide-based hplc assay for rapidly and efficiently assessing glucosylceramide synthase in vivo
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/524f0c7128c14f34911c53c9faa60238
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Incorporation of Fluorescence Ceramide-Based HPLC Assay for Rapidly and Efficiently Assessing Glucosylceramide Synthase In Vivo

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