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

Summary

This protocol describes an anthrone colorimetric method for quantifying glycogen in rat liver and skeletal muscle across different metabolic states.

Abstract

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.

Introduction

Glycogen is a highly branched polysaccharide that serves as a major energy reservoir in skeletal muscle and liver of mammals, and to a lesser extent in other organs such as the brain, kidney, heart, and adipose tissue¹. During feeding, surplus glucose undergoes polymerization or de novo lipogenesis, and is stored as glycogen or lipids. However, during fasting or physical exertion, glycogen undergoes enzymatic degradation, ensuring a constant and rapid supply of glucose to fulfill energy demands. This buffering capacity of glycogen is essential for maintaining glycemic homeostasis during physiological fast–refeeding cycles

Protocol

All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC; #1905019) at Nanjing Medical University.

1. Preparation of Reagents

1. Refer to the Table of Materials for all reagents used in this study.
2. Prepare all solutions before the onset of experiments.
3. Scale solution volumes as needed.
4. Prepare 1 M sodium hydroxide (NaOH) solution
   1. Weigh 40 g of NaOH pellets.
   2. Add the NaOH pellets to 800 mL distilled water.
   3. Stir the solution with a glass rod until no undissolved particles remain at the base of the vess....

Results

Hepatic and Skeletal Muscle Glycogen Levels Under Different Nutritional States

Overnight fasting significantly decreased blood glucose levels (mean ± standard error of the mean [SEM]) from 7.9 ± 0.4 mM in ad libitum-fed rats to 5.4 ± 0.3 mM. Blood glucose levels were restored to 8.3 ± 0.1 mM after 2 h of refeeding (Figure 3D). Consistent with these changes, hepatic glycogen levels (mean ± SEM) were 45.05 ± 3.85 mg/g tissue under fed condi.......

Discussion

In this article, we describe a modified anthrone colorimetric method for quantifying glycogen in animal tissues. In addition to the conventional sequential steps of tissue harvest, alkaline digestion, glycogen precipitation, alcohol washing, and anthrone-based color development, we incorporated a deproteinization step to reduce background interference (Figure 1). We then applied this method to evaluate changes in glycogen content in the liver and gastrocnemius muscle under distinct physiolog.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tubes	LABGIC	BS-15-M	Used for sample processing and reaction setup
15 mL microcentrifuge tubes	LABGIC	BS-150-M-S	Used for lysate handling and precipitation steps
20X TBST	Solarbio Life Sciences	T1082	Buffer for membrane washing in western blot
37 oC Incubator	Shanghai Jinghong Instrument	HWS-150	BCA assay plate incubation
50 mL centrifuge tubes	LABGIC	CT-112-50A	Used for tissue digestion and extraction
96-well plate	LABGIC	BS-MP-96W-CL	Plate-based absorbance measurement
Analytical balance	Changzhou Xingyun	FA2204N	Weigh chemical powders
Anthrone	Shanghai Aladdin Biochemical Technology	A108571	Reagent for glycogen colorimetric detection
Anti-GYS1 antibody	Proteintech	10566-1-AP	Detection of glycogen synthase 1
Anti-GYS2 antibody	Proteintech	22371-1-AP	Detection of glycogen synthase 2
Anti-phospho-GYS1/2 (Ser641) antibody	Cell Signaling Technology	47043S	Detection of phosphorylated GYS
Anti-phospho-PYGL (Ser15) antibody	Abcam	ab227043	Detection of phosphorylated glycogen phosphorylase
Anti-PYGL antibody	Proteintech	15851-1-AP	Detection of liver glycogen phosphorylase
Anti-PYGM antibody	Novus Biologicals	NBP2-16689	Detection of muscle glycogen phosphorylase
BCA assay kit	Beyotime	P0012	Protein quantification
Benzoic acid	Shanghai Aladdin Biochemical Technology	B116255	Preparation of saturated solution for standards
Blunt forcepts	Shanghai Jinzhong	J42010	Toe pinching; tissue dissection
BSA Fraction V	Beyotime	ST023	Blocking agent in western blot
Chemiluminescent imager	Tanon	Tanon 5200 Multi	Detection of western blot signals
Chemiluminescent substrate (ECL)	Beyotime	P0018S	Signal development in western blot
Distilled water	Beyotime	ST872	Solvent for reagent preparation
Epinephrine hydrochloride	Sigma-Aldrich	E4642	Induction of glycogen breakdown in vivo
Ethanol	Macklin	E809065	Glycogen precipitation and washing
Fine surgical scissors	Shanghai Jinzhong	JA2601	Tissue dissection
Fresco 17 centrifuge	Thermo Fisher Scientific	75002402	Sample centrifugation
Glucometer	i-SENS	CareSens N	Measurement of blood glucose
Glucometer	i-SENS	CareSens N	Measure tail vein blood glucose level
Glucometer strips	i-SENS	CareSens N Strips	Measure tail vein blood glucose level
Glucose	Macklin	G6172	Preparation of standard curve
Glycogen stock	Beyotime	D0812	Preparing a series of glycogen standards
Goat anti-rabbit IgG secondary antibody	Thermo Fisher Scientific	31462	Detection of primary antibodies
Handheld homogenizer	GreenPrima	PB100	Tissue homogenization
India ink	Sigma-Aldrich	198285	Total protein staining for normalization
Isoflurane	RWD Life Science	R510-22-10	Animal euthanasia
Laboratory heating block	Changzhou Guowang	GWJ300-1	Sample heating during assays
Lithium chloride	Macklin	767397	Enhances glycogen precipitation
Methanol	Shanghai Aladdin Biochemical Technology	M116115	Glycogen washing step
Microplate reader	BioTek	ELx800	Measurement of absorbance at 620 nm
Nitrocellulose membrane	Millipore	HATF04700	Protein transfer membrane
Orbital shaker	DLAB SCIENTIFIC CO.,LTD	SK-O180-S	chemiluminescent substrate incubation
P1000 Pipettor	Thermo Fisher Scientific	4641100N	Transfer solution
P200 Pipettor	Thermo Fisher Scientific	4641080N	Residual liquid removal
Phosphatase inhibitors (PhosSTOP)	Roche	4906845001	Preservation of protein phosphorylation
Plastic containers (for tissue storage)	Thermo Fisher Scientific	6A0008	Storage of harvested tissues
Protease inhibitors cocktail	MCE	HY-K0011	Prevention of protein degradation
RIPA lysis buffer	Beyotime	P0013B	Protein extraction
SDS-PAGE gel kit	Beyotime	P0012A	Protein separation
SDS-PAGE loading buffer (6X)	Beyotime	P0015F	Mix with protien samples before loading into SDS-PAGE gel wells
SDS-PAGE running buffer (5x)	Beyotime	P0014D	SDS-PAGE electrophoresis
Small Animal Anesthesia Machine	RWD Life Science	R500	Animal euthanasia
Sodium hydroxide	Macklin	S817977	Tissue digestion and glycogen extraction
Sprague-Dawley rats	Charles River Laboratories	Crl: CD(SD)	Experimental animal model
Spray bottle	Beyotime	TB6310	Apply 75% ethanol to animal fur for wetting and disinfection
Standard chow diet	Jiangsu-Xietong, Inc.	XTI01ZJ-009	Rodent maintenance diet
Surgical scissors	Shanghai Jinzhong	J22010	Tissue dissection
Syringe (sterile) with 25-gauge needle	Beyotime	FS801	Intraperitoneal injection and tail vein pricking
Thiourea	Shanghai Aladdin Biochemical Technology	T112512	Stabilizes anthrone reagent
Transfer buffer (10x)	Solarbio Life Sciences	D1060	Protein transfer onto membrane
Trichloroacetic acid	Shanghai Yuanye Bio-Technology	W11293	Protein precipitation
Volumetric flasks (100 mL, 1 L)	Shanghai Aladdin Biochemical Technology	V2853	Preparation of accurate solution volumes
Vortex	DLAB SCIENTIFIC CO.,LTD	8031102000	Sample mixing
Water bath	Jinghong	DK-S26	Heating samples for reactions