Radiochemical Assessment of Glycogen Synthase Enzyme Activity in Animal Tissue

Summary

The animal tissue assay utilizes nuclear scintillation detection to determine the enzymatic activity of glycogen synthase by measuring the incorporation of radio-labeled glucose into glycogen.

Abstract

Glycogen is an important metabolite to many eukaryotic cells, integral to energy storage and quick energy release. The building of the molecule is dependent on glycogen synthase, a highly regulated enzyme with the primary function of facilitating the sequential attachment of glucose units in a growing glycogen polymer. This enzyme is allosterically activated by glucose-6-phosphate and inhibited at multiple sites by phosphorylation. Knowledge of glycogen synthase enzymatic activity is a key component of understanding glycogen metabolism, muscle energy levels, and metabolic disorders such as diabetes and glycogen storage diseases. Described here is a highly sensitive and effective method of determining the activity of glycogen synthase in animal muscle tissue by measuring the amount of integrated glucose, from radio-labeled [14C]-UDP-glucose, into glycogen molecules. This procedure utilizes radioactive ¹⁴C and will require the proper authorization and lab space for use and disposal. This procedure will also require access to a liquid scintillation counter.

Introduction

Glycogen, a highly branched polysaccharide composed of glucose units, serves as an important energy reserve in various tissues, including muscles, liver, and brain¹. Its synthesis and degradation are orchestrated by a complex system of specialized enzymes and regulatory proteins. Vital to its biosynthesis is the enzyme glycogen synthase (GYS), of which there are two isoforms (GYS1 and GYS2). GYS1 is a regulatory enzyme directly responsible for the creation of glycogen from glucose in non-liver cells, while GYS2 functions in the liver. This process, known as glycogenesis, is the sequential addition of an individual unit of glucose onto a growing glycogen chain. GYS catalyzes the transfer of glucose from the intermediate uridine diphosphate glucose (UDP-glucose) to the end of a glycogen chain, creating an alpha-1,4-glycosidic linkage². In coordination with the glycogen branching enzyme, this reaction extends glycogen molecules, providing both efficient energy storage and release.

Assay techniques exist to measure the activity of the enzyme, with one of the more common groupings of methods consisting of those of the spectrophotometric variety^3,4. These assays typically rely on a system of indirect coupled reactions and measure the absorbance of the downstream product, NADH, at 340 nm. One unit of NADH is consumed for every unit of UDP freed from UDP-glucose by glycogen synthase, producing a measurable excitation that directly correlates to glycogen synthase activity^5,6. While spectrophotometry assays are relatively inexpensive and accessible, they come with the minor downside of relying on an indirect reaction and major downsides of interference by sample turbidity and lower sensitivity, especially an issue for smaller samples or samples with little/highly phosphorylated glycogen synthase. Fluorometric assays, using the coupled reactions described above, have been developed to improve sensitivity in muscle tissue from human⁷ and mouse⁸. Other techniques exist for quantifying glycogen synthase, such as immunoblotting⁹, and immunohistochemistry¹⁰; however, these techniques are only able to quantify the physical amount of enzyme and, at best, can infer activity from phosphorylation but not directly quantify enzyme activity¹¹.

Among these enzyme assay techniques, radiochemical methods remain the most widely used for determining glycogen synthase activity in mammalian tissue due to their lack of interference by sample turbidity and their high sensitivity^8,9. These assays incorporate radiolabeled glucose from UDP-glucose labeled with either 14C¹² or 3H¹³ into glycogen, allowing for direct measurement of enzyme activity over a set amount of time. In the following procedure, a tissue sample containing glycogen synthase is exposed to UDP-glucose and glycogen primer. Glycogen synthase catalyzes the reaction shown in the equation below:

UDP-[¹⁴C]glucose + (glycogen)_n → (glycogen)_n+1[14C] + UDP

After a set amount of time, the reaction is terminated and the glycogen containing radiolabeled glucose is either precipitated onto filter paper¹² (as described below) or subjected to gel filtration as described by Niederwanger et al.¹⁴ and measured using a scintillation detector. This method also includes a separate identical reaction that includes glucose-6-phosphate (G6P), an allosteric activator of glycogen synthase. Under conditions with sufficient levels of G6P, glycogen synthase activity is maximally activated, showcasing the absolute potential for glycogen synthase activity of a given sample. The method described here was modified from Suzuki and colleagues¹⁵ to a 96-well format and is optimized for use in mouse muscle tissue; however, the protocol can be altered for other tissues and animal species.

Despite the inherent cost and safety considerations associated with radioactive materials, radiochemical assays offer significant advantages over non-radioactive alternatives, provided instruments are available. The following procedure details a highly sensitive and robust means of determining the activity of glycogen synthase, a key enzyme in glycogen metabolism, in animal muscle tissue.

Protocol

All procedures were approved by the Ball State University Animal Care and Use Committee. Male and female cohorts of B6;129-Gaa^tm1Rabn/J wildtype mice, aged 3 months, are used as a representative example in this manuscript. The reagents and the equipment used are listed in the Table of Materials.

1. Preparation prior to assay

1. Harvest tissue by cervical dislocation¹⁶ and immediately freeze in liquid N2 to preserve glycogen stores and store in liquid N2 or ultralow freezer until the start of the assay.
2. Prepare the stock solutions prior to the start of the glycogen synthase assay (Table 1).
3. Determine the number of replicate mouse samples and controls desired for the assay. Each mouse sample requires four total replicates, two for each reaction (presence or absence of G6P) (see Figure 1 and Table 2).
4. Cut chromatography paper into 4 cm strips. Glycogen attaches to this paper and remains bound during the washing process. Using the width (6 mm) of a paper shredder, shred chromatography paper into thin individual segments.
5. Using a pencil, label designated paper strips with mouse identification, blanks, and totals. Avoid using ink.
6. Load chromatography papers into the wooden block template. Each template holds up to 12 papers, corresponding to one row of a 96-well plate.
7. Obtain chromatography paper clamps (clipboard tops).
8. Prepare one clamp for each row of reaction by clamping the edges of chromatography papers, leaving the majority of papers' surface area exposed. Ensure labeled edges are visible.
9. Label 1.7 mL microfuge tubes with sample identification. Two tubes per sample; one for homogenate and one for supernatant.
10. Prior to the start of the assay, prepare both the + and - G6P reaction mixtures, see Table 3 for more information. Prepare 2,400 µL aliquots of each reaction mixture.

2. Radiochemical glycogen synthase activity reaction

CAUTION: This step contains the use of [14C]-UDP-glucose, which is a radioactive material. Handle, store, and dispose of in a manner approved by the Nuclear Regulatory Commission (NRC).

1. Add 30 µL of [14C]-UDP-glucose (0.1 µCi/ µL) to 2,400 µL aliquots of both (+/- G6P) reaction mixes for a final concentration of 0.0012 µCi/ µL. This recipe is suitable for 18 samples.
   NOTE: The volume of [14C]-UDP-glucose can be changed to conserve usage and ensure adequate sensitivity. For example, use 50 µL for mouse brain tissue and 25 µL for mouse heart.
2. Vortex reaction mixtures and aliquot 50 µL to each well of a 96-well plate that will hold samples, blanks, and totals (Table 2). Afterward, cover with plate film and place at 4 C.
3. Set out the protease inhibitors to thaw at room temperature. These inhibitors are itemized in Table 4 with additional information.
4. Mass out 30-50 mg of frozen tissue. ~30 mg is optimal for skeletal muscle and heart samples; ~50 mg for brain. Keep tissue and microfuge tubes in liquid N2 and quickly weigh tissue to ensure the tissue stays frozen. Use a cooled pestle to grind tissue further.
5. Prepare homogenization buffer with protease inhibitors on ice.
6. One sample at a time, move the tube with tissue from liquid N2 to ice with a cold cap for 1 min. Add a cold cap (tube cap with three small holes cooled in liquid N2) to the sample tube while still in liquid N2. This prevents tissue loss as residual liquid N2 in the tube evaporates. Once out of liquid N2, samples should remain in ice.
7. Once on ice, add volume (10-30 µL/mg of tissue) of homogenization buffer to the sample and homogenize the sample with Tissue-Tearor. After completion of these two steps, move on to the next sample.
   NOTE: Volume of buffer added to samples may be optimized depending on the source of tissue. For example: 30 µL /mg for skeletal muscle, 15 µL /mg for heart, and 10 µL /mg for brain.
8. Centrifuge samples for 5 min at 4 ˚C at 3,600 g and transfer supernatant to a clean microfuge tube. Be careful not to touch the pellet. Vortex and add ~150 µL to corresponding wells in a v-bottom 96-well plate on ice. Save the remainder for supernatant for Bradford assay (see step 2.15)
   NOTE: If desired, additional sample aliquots may be saved for later assays such as immunoblotting.
   CAUTION: Steps 2.9 through 2.14 require handling of ¹⁴C. Dispose of all contaminated materials in a manner approved by the NRC.
9. Remove the reaction mix plate from 4 ˚C and heat to 30 ˚C on the thermocycler with the film removed. During this time, obtain 1 L of -20 ˚C 66% ethanol and add to a wash beaker with a stir rod on stirring plate.
10. Initiate reaction by adding 25 µL of sample supernatant to reaction wells and 25 µL of homogenization buffer to blank and total wells. Use a multichannel pipette to add to 1 row on the reaction plate at a time, waiting for at least 1 min between rows. Record the time that the reaction was initiated for each row. See Table 2 for more information. Use a multichannel pipette and ensure that equal volumes of liquid are added.
11. Allow 10-15 min for the reaction to run; however, run time should be identical between rows.
12. Aspirate 55 µL from each well using a multichannel pipette and dispense onto the respective chromatography papers prepared previously. Mix the reaction by pipetting up and down before aspiration. Spot liquid in the center of the chromatography papers.
13. Remove papers with totals from the clamp using forceps after spotting liquid from the well. These can be laid out on aluminum foil safely to dry. Drop all other chromatography papers into the stirring 66% ethanol bath. Glycogen is insoluble in 66% ethanol, while [14C]-UDP-glucose is soluble and will wash off chromatography papers.
14. Repeat steps 2.11 and 2.12 for each row on the reaction plate. Ensure each row's reaction time is the same by recording the start and stop times of the reaction.
15. Once all rows have been added to the stirring 66% ethanol, wash for 15 min.
    NOTE: During this step and subsequent wash steps, sample aliquots may be prepared for complementary assays, such as the Bradford assay, responsible for determining the total volume of protein for normalization, which is necessary in calculating the enzymatic activity.
16. Dispose of 66% ethanol without losing chromatography papers. Wash with 1 L of new 66% ethanol for 30 min. This 66% ethanol can be at room temperature.
17. Repeat the previous step for a total of three 30 min 66% ethanol washes.
    NOTE: If desired, the 66% ethanol from the final wash step can be saved and stored at -20 ˚C for subsequent assays.
18. After removal of 66% ethanol, submerge all chromatography papers in the wash beaker in acetone. Acetone facilitates the drying process.
19. Remove chromatography papers from acetone and lay them on aluminum foil. Save or dispose of acetone.
20. Dry chromatography papers under a heat lamp for 15 min.
    NOTE: Dry papers may be stored indefinitely. Papers may be stored safely until ready to proceed to scintillation detection.

3. Measuring activity with scintillation detection

1. Using forceps, move chromatography papers into scintillation vials held in scintillation racks. Record how the sample papers are oriented.
2. Add 5 mL of scintillation fluid to each vial using the lid pump. Screw on caps for each vial.
3. Load samples into the scintillation detector and load the halt rack behind the samples. Ensure the program for monitoring ¹⁴C is selected. Start detection by selecting Automatic counting.
4. Print results
5. Dispose of scintillation tubes and contents in a manner approved by the NRC.

Results

Following the protocol, a printout created from the scintillation detector will contain the measured amount of ionizing radiation emitted from each chromatography paper containing vial, directly correlated to the activity level of glycogen synthase. The scintillation detector will compute the counts per minute (CPM) of detected radiation for each sample, blank, and total over a set amount of time. First convert CPM to DPM (disintegrations per min), which is based on the efficiency of the scintillation counter used (DPM=C...

Discussion

Measurement of glycogen synthase enzyme activity is a valuable tool for monitoring glycogen metabolism in various metabolic and disease states. Measurement in the absence and presence of glucose-6-phosphate (G6P) offers insight into the regulatory status of the enzyme and an indirect index of the phosphorylation state. [U-¹⁴C]-UDP-glucose incorporation assay is an efficient and well-documented technique for measuring glycogen synthase enzymatic activity, offering high sensitivity, substrate specificity, and ad...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
[14C] Uridine 5-diphospho-glucose	American Radiolabeled Chemicals	ARC 0154
31 ETCHR chromatography paper	Whatman	3031 915
Benzamidine hydrochloride hydrate	Sigma	B6506
Beta mercaptoethanol	Sigma	M3148
Dry bath	Torey Pines Scientific	SC25
Ethanol (200 proof)	PharmcoAaper	111000200
Ethylene glycol tetraacetic acid (EGTA)	Sigma	E4378
Ethylenediaminetetraacetic acid (EDTA)	Sigma	E5134
Glucose-6-phosphate sodium salt	Sigma	G7879
Glycogen from rabbit liver (Type III)	Sigma	G8876
Microplate, flat-bottom, 96 well	Greiner	655101
Microplate, pcr, 96 well	DOT Scientific	951-PCR
Multi-Purpose Scintillation Counter	Beckman Coulter	LS 6500
Potassium fluoride dihydrate	Sigma	221872
Protease inhibitor cocktail	GoldBio	GB-108-2
Scintillation cocktail	Reasearch Products International	111195
Scintillation vials	Reasearch Products International	125509
Sodium fluoride	Sigma	S7920
Tissue Tearor	Biospec Products	Model 398
Tosyllysine Chloromethyl Ketone (TLCK)	Sigma	T7254
Trizma base (99.9%)	Sigma	T1503
Trizma hydrochloride	Sigma	T3253
Uridine 5-diphospo-glucose	Sigma	U-4625