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

Se-Hyung Park; Robert N Helsley; Leila Noetzli; Ho-Chou Tu; Kristina Wallenius; Gavin O'Mahony; Jeremie Boucher; Jianming Liu; Samir Softic

doi:10.1016/j.xpro.2021.100731

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

Se-Hyung Park, Robert N Helsley, Leila Noetzli, Ho-Chou Tu, Kristina Wallenius, Gavin O'Mahony, Jeremie Boucher, Jianming Liu, Samir Softic

Research output: Contribution to journal › Article › peer-review

4 Scopus citations

Abstract

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).

Original language	English
Pages (from-to)	100731
Journal	STAR Protocols
Volume	2
Issue number	3
DOIs	https://doi.org/10.1016/j.xpro.2021.100731
State	Published - Sep 17 2021

Bibliographical note

Keywords

Animals
Carbohydrate Metabolism
Enzyme Assays/methods
Fructokinases/antagonists & inhibitors
Fructose/metabolism
Hepatocytes/enzymology
Humans
Luminescence
Luminescent Measurements/methods
Mice
Non-alcoholic Fatty Liver Disease

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1016/j.xpro.2021.100731

Cite this

@article{7f6c2578ab0b44c89d6ee97ef00512ed,

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

abstract = "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). ",

keywords = "Animals, Carbohydrate Metabolism, Enzyme Assays/methods, Fructokinases/antagonists & inhibitors, Fructose/metabolism, Hepatocytes/enzymology, Humans, Luminescence, Luminescent Measurements/methods, Mice, Non-alcoholic Fatty Liver Disease",

author = "Se-Hyung Park and Helsley, {Robert N} and Leila Noetzli and Ho-Chou Tu and Kristina Wallenius and Gavin O'Mahony and Jeremie Boucher and Jianming Liu and Samir Softic",

note = "{\textcopyright} 2021 The Author(s).",

year = "2021",

month = sep,

day = "17",

doi = "10.1016/j.xpro.2021.100731",

language = "English",

volume = "2",

pages = "100731",

number = "3",

}

TY - JOUR

T1 - A luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes

AU - Park, Se-Hyung

AU - Helsley, Robert N

AU - Noetzli, Leila

AU - Tu, Ho-Chou

AU - Wallenius, Kristina

AU - O'Mahony, Gavin

AU - Boucher, Jeremie

AU - Liu, Jianming

AU - Softic, Samir

PY - 2021/9/17

Y1 - 2021/9/17

N2 - 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).

AB - 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).

KW - Animals

KW - Carbohydrate Metabolism

KW - Enzyme Assays/methods

KW - Fructokinases/antagonists & inhibitors

KW - Fructose/metabolism

KW - Hepatocytes/enzymology

KW - Humans

KW - Luminescence

KW - Luminescent Measurements/methods

KW - Mice

KW - Non-alcoholic Fatty Liver Disease

U2 - 10.1016/j.xpro.2021.100731

DO - 10.1016/j.xpro.2021.100731

M3 - Article

C2 - 34409309

VL - 2

SP - 100731

JO - STAR Protocols

JF - STAR Protocols

IS - 3

ER -

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

Abstract

Bibliographical note

Keywords

UN SDGs

Access to Document

Fingerprint

Cite this