Chapter 4
Biochemical Measurement of Glycogen: Method
to Investigate the AMPK-Glycogen Relationship
Elite Possik and Arnim Pause
Abstract
Glycogen is a main carbohydrate energy storage primarily found in fungi and animals. It is a glucose
polymer that comprisesα(1-4) glycosidic linkages attaching UDP-glucose molecules linearly andα(1-6)
linkages branching glucose chains every 8‐10 molecules to the main backbone chain. Glycogen synthase,
branching enzyme, and glycogen phosphorylase are key enzymes involved in glycogen synthesis and
degradation. These enzymes are tightly regulated by upstream kinases and phosphatases that respond to
hormonal cues in order to coordinate storage and degradation and meet the cellular and organismal
metabolic needs. The 5’AMP-activated protein kinase (AMPK) is one of the main regulators of glycogen
metabolism. Despite extensive research, the role of AMPK in glycogen synthesis and degradation remains
controversial. Specifically, the level and duration of AMPK activity highly influence the outcome on
glycogen reserves. Here, we describe a rapid and robust protocol to efficiently measure the levels of
glycogen in vitro. We use the commercially available glycogen determination kit to hydrolyze glycogen
into glucose, which is oxidized to formD-gluconic acid and hydrogen peroxide that react with the OxiRed/
Amplex Red probe generating a product that could be detected either in a colorimetric or fluorimetric plate
format. This method is quantitative and could be used to address the role of AMPK in glycogen metabolism
in cells and tissues.
Summary
This chapter provides a quick and reliable biochemical quantitative method to measure glycogen in cells
and tissues. Briefly, this method is based on the degradation of glycogen to glucose, which is then
specifically oxidized to generate a product that reacts with the OxiRed probe with maximum absorbance
at 570 nm. This method is very accurate and highly sensitive. In the notes of this chapter, we shed the light
on important actions that should be followed to get reliable results. We also state advantages and
disadvantages of this method in comparison to other glycogen measurement techniques.
Key wordsGlycogen, Glucose, Colorimetric assay, Hydrolysis enzyme mix, Development enzyme
mix, Oxiprobe, Biochemical titration, Fluorimetric, Colorimetric1 Introduction
Glycogen is a complex carbohydrate that serves as a major energy
storage readily used to meet cellular and organismal metabolic
requirement. It is a multibranched glucose polymer that exists in
fungi and in invertebrate and vertebrate animals [1]. GlucosylDietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_4,©Springer Science+Business Media, LLC 2018
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