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 journalArticlepeer-review

2 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 languageEnglish
Pages (from-to)100731
JournalSTAR Protocols
Volume2
Issue number3
DOIs
StatePublished - Sep 17 2021

Bibliographical note

© 2021 The Author(s).

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

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