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

STAR Protocols (Sep 2021)

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

Affiliations

Se-Hyung Park: Department of Pediatrics, Division of Pediatric Gastroenterology, University of Kentucky College of Medicine, 900 South Limestone, Wethington Rm 527, Lexington, KY 40536, USA
Robert N. Helsley: Department of Pediatrics, Division of Pediatric Gastroenterology, University of Kentucky College of Medicine, 900 South Limestone, Wethington Rm 527, Lexington, KY 40536, USA
Leila Noetzli: Alnylam Pharmaceuticals Inc., Cambridge, MA 021428, USA
Ho-Chou Tu: Alnylam Pharmaceuticals Inc., Cambridge, MA 021428, USA
Kristina Wallenius: Bioscience, Research and Early Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Gavin O’Mahony: Medicinal Chemistry, Research and Early Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Jeremie Boucher: Bioscience, Research and Early Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; The Lundberg Laboratory for Diabetes Research, University of Gothenburg, Gothenburg, Sweden; Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden
Jianming Liu: Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Mölndal, Gothenburg 43183, Sweden; Corresponding author
Samir Softic: Department of Pediatrics, Division of Pediatric Gastroenterology, University of Kentucky College of Medicine, 900 South Limestone, Wethington Rm 527, Lexington, KY 40536, USA; Department of Pharmacology and Nutritional Sciences, University of Kentucky College of Medicine, Lexington, KY 40536, USA; Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard Medical School, Boston, MA, USA; Corresponding author

Journal volume & issue: Vol. 2, no. 3
p. 100731

Abstract

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Summary: 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).

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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