Anthrone Colorimetric Method for Quantifying Glycogen in Rat Liver and Skeletal Muscle

Glycogen is a major intracellular glucose reserve in liver and skeletal muscle and plays a fundamental role in carbohydrate metabolism. Hepatic glycogen is essential for preserving systemic glycemic homeostasis, whereas skeletal muscle glycogen supports physical activity and exercise performance. Accurate quantification of tissue glycogen is therefore critical for studies in which glycogen metabolism is dynamically regulated or pathologically altered. Here, we describe a reliable and sensitive anthrone colorimetric method for quantifying glycogen content in rat liver and skeletal muscle. The protocol consists of sequential steps, including alkaline digestion, deproteinization, glycogen precipitation, alcohol washing, and color development using anthrone reagent. The applicability of this method is demonstrated in rats subjected to three physiologically relevant conditions: ad libitum feeding, overnight fasting, and short-term refeeding. These experimental paradigms capture key metabolic states associated with glycogen storage, depletion, and resynthesis. Orthogonal western blot analysis of key enzymes, glycogen synthase and glycogen phosphorylase, provides mechanistic support to the observed metabolic changes. The applicability of this method is further demonstrated in animals treated with epinephrine to model acute stress-induced glycogen breakdown. Collectively, this method provides a wide dynamic range and sufficient sensitivity to quantify tissue glycogen across markedly different physiological states. It offers a reliable tool for investigating glycogen metabolism in vivo and can also be applied to in vitro cell culture models.