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

STAR Protoc. 2021 Aug 9;2(3):100731. doi: 10.1016/j.xpro.2021.100731. eCollection 2021 Sep 17.

Authors

Se-Hyung Park¹, Robert N Helsley¹, Leila Noetzli², Ho-Chou Tu², Kristina Wallenius³, Gavin O'Mahony⁴, Jeremie Boucher^{3

5

6}, Jianming Liu⁷, Samir Softic^{1

8

9}

Affiliations

¹ Department of Pediatrics, Division of Pediatric Gastroenterology, University of Kentucky College of Medicine, 900 South Limestone, Wethington Rm 527, Lexington, KY 40536, USA.
² Alnylam Pharmaceuticals Inc., Cambridge, MA 021428, USA.
³ Bioscience, Research and Early Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
⁴ Medicinal Chemistry, Research and Early Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
⁵ The Lundberg Laboratory for Diabetes Research, University of Gothenburg, Gothenburg, Sweden.
⁶ Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden.
⁷ Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Mölndal, Gothenburg 43183, Sweden.
⁸ Department of Pharmacology and Nutritional Sciences, University of Kentucky College of Medicine, Lexington, KY 40536, USA.
⁹ Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard Medical School, Boston, MA, USA.

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: Cell Biology; Cell-based assays; Metabolism.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

Fructose
Fructokinases
ketohexokinase