A luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes

Ketohexokinase (KHK) catalyzes the first step of fructose metabolism. Inhibitors of KHK enzymatic activity are being evaluated in clinical trials for the treatment of non-alcoholic fatty liver disease (NAFLD) and diabetes. Here, we present a luminescence-based protocol to quantify KHK activity. The...

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Autores principales: Park, Se-Hyung, Helsley, Robert N., Noetzli, Leila, Tu, Ho-Chou, Wallenius, Kristina, O’Mahony, Gavin, Boucher, Jeremie, Liu, Jianming, Softic, Samir
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2021
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8361265/
https://www.ncbi.nlm.nih.gov/pubmed/34409309
http://dx.doi.org/10.1016/j.xpro.2021.100731
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author Park, Se-Hyung
Helsley, Robert N.
Noetzli, Leila
Tu, Ho-Chou
Wallenius, Kristina
O’Mahony, Gavin
Boucher, Jeremie
Liu, Jianming
Softic, Samir
author_facet Park, Se-Hyung
Helsley, Robert N.
Noetzli, Leila
Tu, Ho-Chou
Wallenius, Kristina
O’Mahony, Gavin
Boucher, Jeremie
Liu, Jianming
Softic, Samir
author_sort Park, Se-Hyung
collection PubMed
description Ketohexokinase (KHK) catalyzes the first step of fructose metabolism. Inhibitors of KHK enzymatic activity are being evaluated in clinical trials for the treatment of non-alcoholic fatty liver disease (NAFLD) and diabetes. Here, we present a luminescence-based protocol to quantify KHK activity. The accuracy of this technique has been validated using knockdown and overexpression of KHK in vivo and in vitro. The specificity of the assay has been verified using 3-O-methyl-D-fructose, a non-metabolizable analog of fructose, heat inactivation of hexokinases, and depletion of potassium. For complete details on the use of this protocol, please refer to Damen et al. (2021).
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spelling pubmed-83612652021-08-17 A luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes Park, Se-Hyung Helsley, Robert N. Noetzli, Leila Tu, Ho-Chou Wallenius, Kristina O’Mahony, Gavin Boucher, Jeremie Liu, Jianming Softic, Samir STAR Protoc Protocol Ketohexokinase (KHK) catalyzes the first step of fructose metabolism. Inhibitors of KHK enzymatic activity are being evaluated in clinical trials for the treatment of non-alcoholic fatty liver disease (NAFLD) and diabetes. Here, we present a luminescence-based protocol to quantify KHK activity. The accuracy of this technique has been validated using knockdown and overexpression of KHK in vivo and in vitro. The specificity of the assay has been verified using 3-O-methyl-D-fructose, a non-metabolizable analog of fructose, heat inactivation of hexokinases, and depletion of potassium. For complete details on the use of this protocol, please refer to Damen et al. (2021). Elsevier 2021-08-09 /pmc/articles/PMC8361265/ /pubmed/34409309 http://dx.doi.org/10.1016/j.xpro.2021.100731 Text en © 2021 The Author(s) 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/).
spellingShingle Protocol
Park, Se-Hyung
Helsley, Robert N.
Noetzli, Leila
Tu, Ho-Chou
Wallenius, Kristina
O’Mahony, Gavin
Boucher, Jeremie
Liu, Jianming
Softic, Samir
A luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes
title A luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes
title_full A luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes
title_fullStr A luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes
title_full_unstemmed A luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes
title_short A luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes
title_sort luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8361265/
https://www.ncbi.nlm.nih.gov/pubmed/34409309
http://dx.doi.org/10.1016/j.xpro.2021.100731
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