AMPK

Elite Possik, Arnim Pause

Glycogen is a main carbohydrate energy storage primarily found in fungi and animals. It is a glucose polymer that comprises α(1-4) glycosidic linkages attaching UDP-glucose molecules linearly and α(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 form D-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.