Increases in glycogenin and glycogenin mRNA accompany glycogen resynthesis in human skeletal muscle

Jane Shearer,1 Rhonda J. Wilson,1 Danielle S. Battram,1 Erik A. Richter,2 Deborah L. Robinson,1 Marica Bakovic,1 and Terry E. Graham1

1Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada; and 2Copenhagen Muscle Research Centre, Institute of Exercise and Sport Sciences, University of Copenhagen, Denmark

Submitted 8 March 2005 ; accepted in final form 20 April 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Glycogenin is the self-glycosylating protein primer that initiates glycogen granule formation. To examine the role of this protein during glycogen resynthesis, eight male subjects exercised to exhaustion on a cycle ergometer at 75% O2 max followed by five 30-s sprints at maximal capacity to further deplete glycogen stores. During recovery, carbohydrate (75 g/h) was supplied to promote rapid glycogen repletion, and muscle biopsies were obtained from the vastus lateralis at 0, 30, 120, and 300 min postexercise. At time 0, no free (deglycosylated) glycogenin was detected in muscle, indicating that all glycogenin was complexed to carbohydrate. Glycogenin activity, a measure of the glycosylating ability of the protein, increased at 30 min and remained elevated for the remainder of the study. Quantitative RT-PCR showed elevated glycogenin mRNA at 120 min followed by increases in protein levels at 300 min. Glycogenin specific activity (glycogenin activity/relative protein content) was also elevated at 120 min. Proglycogen increased at all time points, with the highest rate of resynthesis occurring between 0 and 30 min. In comparison, macroglycogen levels did not significantly increase until 300 min postexercise. Together, these results show that, during recovery from prolonged exhaustive exercise, glycogenin mRNA and protein content and activity increase in muscle. This may facilitate rapid glycogen resynthesis by providing the glycogenin backbone of proglycogen, the major component of glycogen synthesized in early recovery.

granule; proglycogen; macroglycogen; recovery; carbohydrate


GLYCOGENIN (GN-1) is the self-glycosylating protein primer that catalyzes glycogen granule formation in skeletal muscle. It is a substrate, catalyst, and enzyme of granule formation as it adds 7–11 glucose residues to a single tyrosine residue on the protein (3). Following glycosylation, GN-1 acts as a substrate for glycogen synthase that, along with branching enzyme, forms a glycogen granule. In the initial stages of glycogen formation, the granule is small, has low carbohydrate content, and is termed proglycogen (PG). When PG granules grow by the addition of glucose residues, larger, mature glycogen granules are formed and are termed macroglycogen (MG). These granules contain the same amount of protein but a larger proportion of carbohydrate, up to five times more carbohydrate than the largest PG molecule (2, 4, 22, 23). Studies have shown PG to be the more dynamic pool of glycogen, as it is often more readily degraded during exercise and is the form that is predominantly synthesized during the early phases of recovery (2, 6, 15).

Under resting conditions, no free deglycosylated GN-1 is present in skeletal muscle; it is all complexed to glycogen granules (30). Upon glycogenolysis, induced by exercise or treatment with pharmacological concentrations of epinephrine, there is a decline in the activity of the protein (29) and translocation from the glycogen/sarcovesicular fraction to the supernatant (30). These results suggest that GN-1 may be inactivated once glycogen granules are degraded. Given this, glycogen resynthesis likely involves 1) the synthesis of new GN-1 protein or 2) the addition of glucose residues to existing glycogen structures. Biochemical and electron microscopic data show both mechanisms to be involved, as there is an increase in granule size and number with glycogen resynthesis (11, 21). New glycogen granule formation would theoretically require the synthesis of new GN-1 protein. Indeed, there are several reports of increases in GN-1 mRNA in response to dietary and exercise regimens that deplete glycogen stores (5, 17). Kraniou et al. (17) demonstrated skeletal muscle GN-1 mRNA to increase 2.8-fold at 3 h postexercise in humans after moderate-intensity exercise. This increase in mRNA occurred despite only marginally lowered levels of glycogen and indicates a rapid increase in gene expression, but not necessarily levels of the protein. Although these results show GN-1 mRNA to be increased with exercise, they do not clearly define the role of GN-1 in glycogen granule synthesis.

The aim of the present study was to examine GN-1 and its role in glycogen resynthesis following an exhaustive exercise bout. By examining the timing and extent of GN-1 gene expression (mRNA), and GN-1 protein in relation to PG and MG, the role of this protein in glycogen synthesis can be elucidated. It is hypothesized that GN-1 gene transcription (mRNA) would occur following glycogen-depleting exercise and that this signal would be associated with increases in GN-1 protein. These changes are expected to occur predominantly in the most rapid phase of PG synthesis and to be associated with new glycogen granule formation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects

Eight male subjects, age 24 ± 2 yr, height 177 ± 6 cm, weight 73 ± 4 kg, (O2 max) 62 ± 7 ml·kg–1·min–1 (means ± SE), volunteered for the study. The study received approval from the Human Ethics Committee of the University of Guelph. Subjects were informed of potential risks involved with the procedure, and consent was obtained. Participants were habitually active, exercising two to three times a week, but they refrained from exercise three days before the experimental protocol.

Pretrial Procedures

At least 1 wk after performing an incremental O2 max test, subjects returned to the laboratory to be familiarized to the experiment and perform a 45-min practice ride at 75% of their O2 max and 3, 30 s sprints at 130% O2 max. This also served to ensure that calculated exercise intensities were correct.

Experimental Protocol

On the day of the experiment, subjects consumed a high-carbohydrate breakfast ≥2 h before the exercise trial (524 ± 74.9 kcal, 76 ± 6% kcal derived from carbohydrates). Subjects exercised to voluntary exhaustion at 75% O2 max on a cycle ergometer. Once exhaustion was reached (107 ± 7.5 min), subjects had a 10-min rest break during which one leg was prepared for muscle biopsy. In an attempt to further lower the muscle glycogen concentration, subjects then returned to the bike and performed five 30-s sprints at 130% O2 max separated by 1-min rest intervals. Not all subjects could complete the sprints, although all sprints were attempted. Immediately after the last sprint, a muscle biopsy and blood sample were obtained (time 0). Muscle biopsies were obtained from the vastus lateralis muscle of each leg with a percutaneous needle biopsy technique under local anesthesia. Every hour, starting at time 0, subjects were given 75 g of carbohydrate (Gatorlode) to facilitate repletion of glycogen stores. Additional muscle biopsies and blood samples were obtained at 30, 120, and 300 min postexercise.

Analyses

Muscle biopsies were rapidly frozen in liquid nitrogen and stored at –80°C until further analysis. GN-1 protein was detected by two methods. In the first method, the ability of the protein to glycosylate or its "activity" was determined. This is an indirect measurement of active GN-1 protein and is proportional to its concentration (29). In the second, glycogenin was detected by immunohistochemistry, a method that allows quantification of protein content regardless of activity. Throughout the remainder of the paper, these two types of glycogenin quantification will be referred to as "activity" and "protein content," respectively.

The priority of analysis was as follows: PG, MG, and GN-1 activity, GN-1 mRNA, and GN-1 protein concentration. Of 32 muscle biopsies (n = 8 samples per time point), all were analyzed for PG and MG, whereas 30 samples (n = 7–8 samples per time point) were analyzed for GN-1 activity and GN-1 mRNA. GN-1 protein concentration was determined on 20 muscle samples (n = 7, 5, 5, and 3 for 0, 30, 120, and 300 min, respectively).

PG and MG. Samples (~10 mg wet wt) were freeze dried and dissected free of visible nonmuscular components, connective tissue, and blood. PG and MG in a 2- to 3-mg (dry wt) portion were analyzed enzymatically as previously described and are reported in millimoles glucosyl units per kilogram dry weight (1, 2). Briefly, ice-cooled 1.5 M PCA (200 µl) was added to 1.5–3.0 mg of freeze-dried muscle samples in 5-ml Pyrex tubes. The muscle was pressed against the glass tubes with a plastic rod to ensure that all the muscle was exposed to acid. The extraction continued on ice for 20 min. The samples were centrifuged at 3,000 rpm for 15 min, after which 100 µl of the PCA supernatant were removed, placed in Pyrex tubes, and used for the determination of MG. The remaining PCA was discarded, and the pellet was kept for the determination of PG. One milliliter of 1 M HCl was added to the MG and to the PG sample; the former was vortexed, whereas the pellet of the latter was pressed against the glass with a plastic rod. The tube weights were then recorded. The tubes were sealed with fitted glass stoppers, and all of the samples were placed in the water bath (100°C) for 2 h, after which the tubes were reweighed, and any change of >50 µl was rectified with the addition of deionized water. The samples were then neutralized with 2 M Trizma base, vortexed, centrifuged at 3,000 rpm for 5 min, and transferred to Eppendorf tubes for analysis of glucosyl units by use of the method of Bergmeyer (7) or stored at –80°C. Total glycogen (Gt) is the sum of the PG and MG values. Net rates of glycogen synthesis are calculated as the difference in glycogen concentration between two time points divided by time and are expressed in millimoles glucosyl units per kilogram dry weight per minute.

RNA isolation. Total RNA was extracted from 30 mg wet wt of muscle by a modified Chomczynski and Sacchi (8) method using TRIzol reagent (GIBCO-BRL). Briefly, 1 ml of TRIzol reagent was added, and tissues were homogenized for 30 s (Powergen 125, Fischer). Homogenized samples were incubated for 3 min at room temperature before the addition of 0.2 ml of chloroform. Samples were shaken by hand and allowed to sit for 5 min at room temperature before being centrifuged for 15 min at 12,000 g. The higher RNA water layer was removed, and 0.5 ml isopropyl alcohol was added to precipitate RNA. Samples were precipitated at 4°C for 30 min and then centrifuged at 12,000 g for 30 min. Pellets containing total RNA were washed with 2x 0.5 ml of ethanol, air dried, and then resuspended in 20 µl of RNAase-free water. Concentration and purity of the RNA isolation were determined on 1 µl of the extract by spectroscopy.

Reverse transcription. Reverse transcription (RT) of samples was performed using the Thermoscript RT-PCR System (GICBO-BRL). Oligo(dT)20 primers (1 µl) were combined with 1 µg of total RNA and diethyl pyrocarbonate (DEPC)-treated water (to 10 µl). RNA was denatured by heating for 5 min at 65°C before addition of 10 µl of a reaction mixture containing 100 mM Tris acetate (pH 8.4), 150 mM potassium acetate, 16 mM magnesium acetate, 10 mM DTT, 4 U RnaseOUT (GIBCO-BRL), 1 mM dNTP mix, 1.5 U reverse transcriptase (Thermoscript, GICBO-BRL), and 1 µl of DEPC water. Samples were gently mixed and then transferred to a thermocycler (Techgene, Cambridge, MA), where samples were heated to 55°C for 45 min, and then 85°C for 5 min to terminate the RT reaction. Rnase H (1 µl; GICBO-BRL) was added, and samples were incubated at 37°C for 20 min before being stored at –20°C until further analysis.

PCR. Prior to PCR of experimental samples, optimal, nonsaturating conditions for PCR were established (annealing temperature, no. of cycles, MgCl2 concentration). All samples from a given subject were run simultaneously. mRNA content of genes was determined in duplicate by PCR. The PCR reaction mixture was 50 µl [RT reaction, 20 mM Tris·HCl, pH 8.4, 50 mM KCl, 0.2 mM each of dCTP, dATP, dGTP, and dTTP, 1.5 mM MgCl2, 0.5 mM each of forward and reverse primers, and 1.25 U Taq DNA polymerase (GIBCO-BRL)]. PCR products were separated on 2.5% agarose gels containing ethidium bromide by electrophoresis. Gels were visualized by exposure to UV light and documented by an integrating camera and a gel analysis program (Northern Exposure). Software (Image J, National Institutes of Health) was then used to quantify the PCR products. To correct for differences in mRNA qualities, {beta}-Actin mRNA was also amplified and used as a control in each PCR reaction. Forward and reverse {beta}-actin primers were 5'-CCCAAGGCCAACCGCGAGAGAT-3' and 5'-GTCCCGGCCAGCCAGGTCCAG-3' and resulted in a 219-bp product. The PCR cycle profile for {beta}-actin was as follows: 94°C for 2 min, (94°C for 30 s, 62°C for 50 s, 72 for 50 s) x 15 cycles; (94°C for 30 s, 62°C for 50 s, 72°C for 90 s) x 5 cycles, 72°C for 5 min. GN-1 mRNA was quantified using 5'-ACAGCACAGGACCACCAGGA-3' and 5'-GCTCAGAAGCAAGATGCAAC-3' as the forward and reverse primers, respectively. With an annealing temperature of 58°C and 1.5 mM MgCl2, the PCR product was 386 bp. The PCR cycle profile for GN-1 was as follows: 94°C for 2 min, (94°C for 30 s, 58°C for 50 s, 72 for 50 s) x 20 cycles, (94°C for 30 s, 58°C for 50 s, 72°C for 90 s) x 5 cycles, 72°C for 5 min.

Sequencing. To confirm that the expected genes were amplified, the gel-purified (GIBCO-BRL Concert Nucleic Acid Purification System) PCR products for both {beta}-actin and GN-1 (GIBCO-BRL Concert Nucleic Acid Purification System) were sequenced by an automated sequencer at the University of Guelph Molecular Supercenter (ABI Prism 377).

GN-1 activity. GN-1 activity was measured as previously described (13, 29). The term GN-1 activity refers to the ability of GN-1 to transglucosylate a maltose derivative. Wet muscle was ground with a mortar and pestle under liquid nitrogen and weighed into 30- to 40-mg portions. Samples were homogenized in five volumes of buffer (4 mM EDTA, pH 7, 0.1 mM phenylmethylsulfonyl fluoride, 0.1% 2-mercaptoethanol, and 1 mM benzamide at 4°C) and centrifuged at 4°C at 4,200 g for 35 min. The supernatant [containing the glycogen/sarcovesicular fraction (29)] was retained and the myofibrillar pellet discarded. Protein concentrations were measured (Coomassie Plus Protein Reagent Kit, Pierce). Equal amounts of protein (150 µg) were amylolysed with 10 µg/ml {alpha}-amylase (Sigma) for 1 h at 37°C. The amylolysed sample was incubated in a mixture containing 8 µM UDP-[14C]glucose (300 mCi/mmol, ARC), 17 mM MES (pH 7), 5 mM MnSO4, 0.2 mM n-dodecyl-{beta}-D-maltoside (Sigma), and 50 µl of homogenate. The final volume of the incubation was 60 µl. Glucosylation proceeded for exactly 10 min at 37°C before the reaction was stopped by the addition of 16 µl of 0.1 M EDTA (pH 7). Glucose (20 µl, 10 mM), UDPG (20 µl, 20 mM), and deionized water (84 µl; Milli-Q) were added to the 76 µl of sample to avoid nonspecific binding (200 µl final volume). Total radioactivity was measured in 10 µl of the sample while the remaining sample was passed though prewashed C18 Sep-Pak cartridges (Waters). The cartridges were washed with 16 ml of water, and the 14C-labeled glycosylated DBM was eluted with 3 x 1 ml volumes of methanol. Scintillation fluid (10 ml) was added, and the three fractions were counted (Beckman LS5000). The majority of the radioactivity was eluted in the first two fractions. One unit of activity is defined as 1 nmol of [14C]glucose incorporated into DBM per minute per mg of protein (mU·mg protein–1·min–1) (3). Assay coefficient of variation is 14.6% when performed on independent samples of the same biopsy. This value is compatible with reports that the coefficient of variation in the measurement of glycogen ranges from 7 to 10% (1). GN-1 specific activity was calculated as the ratio of GN-1 activity (mU·mg protein–1·min–1) divided by the amount of relative protein (arbitrary units) for each individual sample.

GN-1 protein level. Frozen muscle biopsies were homogenized and treated as previously described by Hansen et al. (13) and as described above in MATERIALS AND METHODSfor measuring GN-1 activity level. Protein concentrations were measured (Pierce Coomassie Plus Protein Reagent Kit). Equal amounts of protein (150 µg) were amylolysed with 10 µg/ml {alpha}-amylase (Sigma) for 1 h at 37°C. To test for nonglycosylated GN-1, samples were also incubated without amylase. GN-1 protein levels were measured by immunoblotting essentially as described (13). Briefly, proteins were transferred to polyvinylidene fluoride membranes following SDS-PAGE electrophoresis (1 mm, 10% gels) (18). Membranes were blocked overnight in 1% Tris-buffered saline-Tween solution (TBS-T) with 5% BSA (Sigma) and 2% skim milk (Nestle) at 4°C with gentle agitation. Blocked membranes were incubated with guinea pig anti-human GN-1 antibody (kindly donated by Dr. P. Roach, Indiana University, Bloomington, IN) diluted 1:2,000 (1% TBS-T) for 1 h at room temperature. The membranes were washed with 1% TBS-T for 1 h (6–10 min intervals) and were subsequently incubated 1 h at room temperature with goat anti-guinea pig IgG horseradish peroxidase-conjugated antibody (Chemicon) diluted 1:10,000 with blocking solution. GN-1 protein was visualized by enhanced chemiluminescence (Amersham) according to the manufacturer's protocol. Protein samples that were not treated with amylase served as a negative control, whereas His-tagged recombinant human GN-1 (kindly donated by Dr. P. Roach) was used as a positive control. GN-1 was quantified using Scion Image Beta 4.02 (Scion, Frederick, MD), and results were expressed relative to the first biopsy (0 min). Results were normalized by blotting for {alpha}-actin.

Blood glucose and insulin. Blood samples were separated into two aliquots; one 3-ml sample was transferred to a nonheparinized tube, where it was allowed to clot. Serum was then extracted and stored for the measurement of serum insulin. Insulin measurements were determined using the radioimmunoassay method (Coat-a-Count, Diagnostic Products). The second aliquot (100 µl) of whole blood was added to 500 µl of 0.6 M perchloric acid and centrifuged, and the supernatant was stored at –20°C for glucose analysis.

Statistical Analysis

Data are presented as means ± SE. A one-way repeated-measures ANOVA test was used to establish differences in GN-1 activity, GN-1 mRNA expression, and PG, MG, Gt, plasma glucose, and insulin between time points. Significant differences were located by a Tukey post hoc test for these measurements. Data for GN-1 protein content and specific activity were analyzed by one-way ANOVA. Significant differences for this test were determined by Dunn's post hoc analysis. Differences were considered significant at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Muscle Glycogen Concentration and Resynthesis

Concentrations of Gt, PG, and MG for all time points are shown in Fig. 1. At time 0, total glycogen level was 59 ± 12 mmol glucosyl units/kg dry wt. PG was the only form to have a significant increase over the first 2 h, with the most rapid resynthesis occurring in the first 30 min of recovery when it had a net synthesis rate that was approximately four times that of MG (Fig. 2). PG accounted for 77% of Gt at time 0, and this declined to 68% of Gt at 300 min of recovery. At all time points, PG concentration showed a significant increase (P < 0.05) from time 0, whereas MG did not increase until 300 min. PG and MG concentrations were significantly different from each other at all time points (P < 0.05; Fig. 1). Net rates of PG, MG, and Gt resynthesis (mmol glucosyl units·kg dry wt–1·min–1) are depicted in Fig. 2. As expected from previous studies (2, 6), the rate of PG resynthesis exceeded that of MG. The rate of PG synthesis declined between the first period (0–30 min) and subsequent time periods (30–120 and 120–300 min, P < 0.05), whereas the rate of MG synthesis remained unchanged. Gt resynthesis decreased significantly (P < 0.05) between 0–30 and 30–120 min, with resynthesis declining from 2.0 ± 0.5 to 0.6 ± 0.1 mmol glucosyl units·kg dry wt–1·min–1 (P < 0.05).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1. Resynthesis of human skeletal muscle glycogen during recovery from exhaustive exercise. Bars represent proglycogen (PG), macroglycogen (MG), and total glycogen concentration. At 300 min, there were significant increases in all types of glycogen. Data are presented as means ± SE; n = 8 muscle biopsies per time point or 32 samples in total. Within a type of glycogen, bars with different letters indicate significant differences between time intervals (P < 0.05).

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Net rates of PG, MG, and total glycogen synthesis during the time intervals 0–30, 30–120, and 120–300 min in human muscle biopsy samples after exhaustive exercise. Rates of total glycogen synthesis were greatest in the initial period (0–30 min). Values represent means ± SE; n = 8 muscle biopsies per time point or 32 samples in total. *Significant difference (P < 0.05) between PG and MG within a time point.

 
GN-1 Activity

Results of GN-1 activity measurements are summarized in Fig. 3. GN-1 activity ranged from 23 to 137 mU·mg protein–1·min–1, with the lowest activity occurring at time 0. GN-1 activity more than doubled from 0 to 30 min (P < 0.05) when the PG rate was maximal and remained elevated from 30 to 300 min. Muscle glycogen synthase has been shown to translocate to the cytoskeleton when glycogen concentrations decrease (24). To exclude that the low GN-1 activity at 0 min was due to loss of GN-1 in the discarded myofibrillar pellet when muscle lysates were prepared, the pellet and supernatant from 12 independent biopsy samples from a similar study were analyzed for GN-1 protein and activity (R. J. Wilson, unpublished observations). These preliminary data indicate that there was no translocation of GN-1 from the supernatant to the myofibrillar pellet when glycogen concentration was low. The relation between GN-1 activity and PG and MG concentration is depicted in Fig. 4.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3. Glycogenin (GN-1) activity (mU·mg protein–1·min–1) during recovery from exhaustive exercise in human skeletal muscle biopsy samples. After exhaustive exercise, GN-1 activity, a measure of the ability of the protein to glycosylate, was determined. Results show an increase in GN-1 activity as time and GN-1 concentration increase. Data represent means ± SE; n = 7–8 samples per time point, 30 samples in total. *Significant difference (P < 0.05) between time points.

 


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4. GN-1 activity in relation to PG and MG concentration in human muscle biopsy samples obtained after exhaustive exercise. The y-axis shows GN-1 concentration; x-axis depicts GN-1 activity, a measure of the ability of the protein to glycosylate. Results show a positive correlation between activity and both PG and MG. Data represent means ± SE; n = 7–8 samples per time point, 30 samples in total as represented by each point on the graph.

 
GN-1 mRNA and Protein

To test for the presence of deglycosylated GN-1 at time 0, all samples were treated with and without {alpha}-amlyase. GN-1 mRNA for the 30-, 120-, and 300-min biopsies is expressed relative to the first biopsy (0 min) that was set at an arbitrary value of 1 (Fig. 5). Significant increases in GN-1 mRNA occurred at 120 min postexercise, whereas increases in protein were detectable at 300 min (P < 0.05). No GN-1 protein was detected on untreated samples despite ample protein present upon amylase treatment (Fig. 5). GN-1 specific activity, a measurement of the enzyme's activity per unit of protein increased at 120 min compared with 0 (P < 0.05; Fig. 6).



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5. A, top: relative GN-1 mRNA and protein levels after exhaustive exercise in human skeletal muscle. Filled bars, mRNA levels; open bars, protein content as measured by Western blotting. Data represent means ± SE. For mRNA, graphs represent 7–8 samples per time point or 30 muscle samples in total. For GN-1 protein, graphs represent n = 7, 5, 3, and 3 samples per time point for 0, 30, 120, 300 min, respectively, or 20 biopsies in total. *Significant difference from time 0 within a measurement (P < 0.05). Bottom: representative blots of GN-1 mRNA, {beta}-actin mRNA (control), and GN-1 protein content. B: representative Western blot of GN-1 protein treated with and without {alpha}-amylase. Lane 1, untreated sample (time 0); lane 2, GN-1 protein from lane 1 treated with {alpha}-amylase (time 0); lane 3, positive His-tagged recombinant human GN-1 protein (+ control). Results show that GN-1 protein is undetectable when untreated.

 


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 6. Specific GN-1 activity (glycosylating ability) normalized to GN-1 protein content (Western blotting) in human muscle biopsy samples during recovery. Data represent GN-1 activity (mU·mg protein–1·min–1) divided by relative protein from Western blotting and are presented as means ± SE; n = 7, 5, 3, and 3 samples per time point for 0, 30, 120, and 300 min, respectively, or 20 biopsies in total. *Significant difference from time 0 (P < 0.05).

 
Sequencing

Sequencing of GN-1 and {alpha}-actin bands obtained from gels confirmed that they were expected products (data not shown).

Blood Glucose and Insulin

Blood glucose was 3.42 ± 0.18 mM following exercise (0 min) and only increased to 5.15 ± 0.33 mM at 30 min despite repeated ingestion of carbohydrate every hour. At 120 and 300 min postexercise, blood glucose levels were 4.92 ± 0.25 and 3.59 ± 0.20 mM, respectively (P < 0.05). Insulin levels were low at exhaustion (1.9 ± 0.37 µU/ml) and peaked at 120 min, when mean insulin values were 31.6 ± 6.4 µU/ml (P < 0.05).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
During glycogen resynthesis, there are two ways to augment glycogen stores: increase the size of existing glycogen granules or initiate the formation of new glycogen granules. In the first scenario, the number of granules in skeletal muscle remains constant as existing granules grow larger, requiring no new GN-1 protein. Such a change would ultimately be accompanied by declines in PG and an increase in MG. Conversely, when new glycogen granules are formed, it would result in an increase in PG and a slow increase or no change in MG and would require additional, functional GN-1 protein. Here, we present data to support the second hypothesis, that during the early phases of glycogen repletion new (PG) granules are formed. This process is likely initiated during exercise itself and is facilitated by the upregulation of both GN-1 mRNA and protein. Only when glycogen granule numbers reach a critical threshold mass do glycogen granules appear to get larger or make the transition from PG to MG. These results provide new insight into the mechanism of glycogen granule formation and repletion in skeletal muscle in the postexercise period.

In the present study, GN-1 mRNA levels more than doubled within 2 h postexercise. Although we cannot distinguish whether these changes resulted from increases in the rate of gene transcription or mRNA stability, the 70% increase in GN-1 protein levels at 300 min postexercise suggests that a portion of the GN-1 mRNA was translated into functional protein. Further evidence of new GN-1 protein synthesis lies in the measurement of GN-1 activity, a measure of the protein's self-glycosylating ability. Results show GN-1 activity to double at 30 min compared with levels seen at exhaustion and to remain elevated for the duration of the study. GN-1 mRNA levels likely increase to replenish GN-1 stores, as the protein appears to be inactivated or degraded upon glycogen granule catabolism (29, 30). This observation was substantiated by the absence of deglycosylated GN-1 at exhaustion. If deglycosylated GN-1 were present, then it would most likely appear at this time point when glycogen concentration and granule numbers were at their lowest. Although it may appear to be a wasteful biological strategy to destroy GN-1 and shortly afterward resynthesize it, it could be problematic to have a free, self-glycosylating protein present in skeletal muscle during exercise, competing with glycolysis for glucose residues.

Although changes in GN-1 mRNA seen in this study are not as large as some other metabolic genes in the postexercise period (26), increases in GN-1 protein would not have to be large to facilitate large gains in glycogen concentration. A small number of GN-1 molecules could store a large amount of glycogen, especially if the granules increase to MG that can contain up to 55,000 glucosyl residues per granule (22, 23). To date, little is known about the regulation of GN-1 gene transcription, although examination of the GN-1 gene shows that it contains several binding sites for developmental, cell-type-specific and muscle-specific transcription factors in its promoter region (31). However, it is clear that there is coordinate regulation of genes involved in metabolism in response to exercise-induced glycogen depletion. Increases in the mRNA of GLUT4, hexokinase II, interleukin-6, citrate synthase, pyruvate dehydrogenase kinase-4 (PDK-4), uncoupling protein-3 (UCP3), fatty acid translocase/CD36, and plasma membrane fatty acid-binding protein all occur during or within 4 h postexercise (16, 17, 25, 27). In addition, levels of mRNA for some of these proteins appear to vary with glycogen concentration, suggesting that glycogen itself or some related protein may promote metabolic gene transcription (25). These findings suggest that there may be a factor released/activated upon glycogen granule degradation that coordinately regulates genes involved in carbohydrate and lipid metabolism. The nature of this factor or signal has yet to be elucidated, however, one such factor may be the AMP-activated protein kinase, the activity of which has been shown to be glycogen dependent (9, 12, 32) and able to activate gene transcription (10, 14, 33).

The sequential creation of new glycogen granules is supported by the analysis of PG and MG in the present study. PG and MG resynthesis during the first 30 min of recovery demonstrated the net rate of PG resynthesis to be four times that of MG. From 30 to 120 min, PG was synthesized 1.7 times faster than MG, whereas in the last time period (120–300 min) PG and MG synthesis were much more modest and very comparable (0.33 for PG and 0.32 mmol glucosyl units·kg dry wt–1·min–1 for MG). Therefore, it appears that the early and rapid increase in PG reflects new glycogen granule formation, whereas the latter increase in MG may represent a slower replenishment of the outer tiers of existing molecules or the transition of PG to MG. These results are consistent with previous reports showing that PG is the predominant form of glycogen synthesized early in recovery (2, 6, 15). Using biochemical measurements of PG and MG in human skeletal muscle, we have shown that PG accumulation is the initial event during glycogen resynthesis (0–4 h) followed by increases in MG (4–24 h) (2). These data are consistent with the findings of Elsner et al. (11), who have demonstrated using radiolabeled glucose a doubling of glycogen concentration in the early phases of glycogen resynthesis yet no increase in glycogen granule size in cultured rat myotubes. These findings have also been confirmed visually using transmission electron microscopy (TEM), a technique that allows the quantification of granule size, number, and subcellular distribution (20). Specifically, results show a 90 and 186% increase in glycogen granule number (0–4 h) in the subsarcolemmal and myofibrillar areas, yet a nonsignificant increase in granule size. In the subsequent time period (4–24 h), the opposite trend was observed, with no increase in granule number yet a large augmentation (160%) of glycogen granule size. Taken together, the results of this and previous studies clearly demonstrate that glycogen resynthesis is a highly ordered, sequential process that involves new glycogen granule formation followed by a transition to making existing granules larger.

The finding that GN-1 activity was elevated from 0 to 30 min of recovery whereas GN-1 mRNA and protein content had not yet increased suggests that GN-1 activity may be regulated by covalent or other modifications. Recent work suggests that both protein-protein dimerization and accessory proteins may be involved. Work in cell culture by Lin et al. (19) has shown that GN-1 catalyzes glycosylation by an intersubunit reaction between two GN-1 molecules as well as an independent, singular protein. This interaction may be a point of regulation, as there is a significant decrease in the self-glycosylating ability of GN-1 when concentrations are low (19). At the lowest GN-1 levels seen in the present study (time 0), glycogen resynthesis occurred rapidly, suggesting that there was enough protein available for intersubunit interactions. Additional regulation of GN-1 may be imparted by the recently discovered GN-1 interacting proteins (GNIP). These small proteins are expressed in skeletal muscle and stimulate GN-1 glycoslyation. Although the roles of GNIP are unclear, they may be involved in the regulation of glycogen granule initiation (28). Specifically, they may influence the ability of GN-1 to glycosylate by imparting conformational changes in the protein, thus imposing an additional site(s) of regulation.

By examining GN-1 mRNA, protein, and activity in conjunction with PG and MG concentrations, this study provides novel data regarding glycogen granule formation in skeletal muscle. The results show that, during recovery from prolonged exhaustive exercise, GN-1 mRNA, protein content, and activity increase in skeletal muscle in a similar time frame to other metabolic genes. Together, these events may facilitate rapid glycogen resynthesis by providing the GN-1 backbone of PG, the major component of glycogen synthesized in early recovery.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by The Natural Sciences and Engineering Research Council of Canada (NSERC), Gatorade Sport Science Institute, the Copenhagen Muscle Research Centre, the Danish Science and Medical Research Council, the Novo-Nordisk Foundation, and the Danish Diabetes Foundation. J. Shearer was supported by an Industrial NSERC scholarship sponsored by Gatorade Sport Science Institute.


    ACKNOWLEDGMENTS
 
The technical assistance Lori Knoll and Premila Sathasivam is much appreciated. We also acknowledge the contributions of Dr. Mark Tarnopolsky (McMaster University), whose laboratory was instrumental in establishing techniques for the quantification of the glycogenin mRNA. Antibodies for the detection of glycogenin were kindly donated by Dr. Peter Roach from Indiana University.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Shearer, Faculty of Medicine, Univ. of Calgary, Rm. 2502, 3330 Hospital Dr. NW, Calgary T2N 4N1, Canada (e-mail: jshearer{at}ucalgary.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adamo KB and Graham TE. Comparison of traditional measurements with macroglycogen and proglycogen analysis of muscle glycogen. J Appl Physiol 84: 908–913, 1998.[Abstract/Free Full Text]
  2. Adamo KB, Tarnopolsky MA, and Graham TE. Dietary carbohydrate and postexercise synthesis of proglycogen and macroglycogen in human skeletal muscle. Am J Physiol Endocrinol Metab 275: E229–E234, 1998.[Abstract]
  3. Alonso MD, Lomako J, Lomako WM, and Whelan WJ. Catalytic activities of glycogenin additional to autocatalytic self-glucosylation. J Biol Chem 270: 15315–15319, 1995.[Abstract/Free Full Text]
  4. Alonso MD, Lomako J, Lomako WM, and Whelan WJ. A new look at the biogenesis of glycogen. FASEB J 9: 1126–1137, 1995.[Abstract/Free Full Text]
  5. Arkinstall MJ, Tunstall RJ, Cameron-Smith D, and Hawley JA. Regulation of metabolic genes in human skeletal muscle by short-term exercise and diet manipulation. Am J Physiol Endocrinol Metab 287: E25–E31, 2004.[Abstract/Free Full Text]
  6. Battram DS, Shearer J, Robinson D, and Graham TE. Caffeine ingestion does not impede the resynthesis of proglycogen and macroglycogen after prolonged exercise and carbohydrate supplementation in humans. J Appl Physiol 96: 943–950, 2004.[Abstract/Free Full Text]
  7. Bergmeyer HU, Bernt K, Schmidt F, and Stork H. D-Glucose determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Methods of Enzymatic Analysis, edited by Bergmeyer HU. New York: Academic, 1974, p. 1196–1201.
  8. Chomcznyski P and Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[CrossRef][ISI][Medline]
  9. Derave W, Ai H, Ihlemann J, Witters LA, Kristiansen S, Richter EA, and Ploug T. Dissociation of AMP-activated protein kinase activation and glucose transport in contracting slow-twitch muscle. Diabetes 49: 1281–1287, 2000.[Abstract]
  10. Durante PE, Mustard KJ, Park SH, Winder WW, and Hardie DG. Effects of endurance training on activity and expression of AMP-activated protein kinase isoforms in rat muscles. Am J Physiol Endocrinol Metab 283: E178–E186, 2002.[Abstract/Free Full Text]
  11. Elsner P, Quistorff B, Hansen GH, and Grunnet N. Partly ordered synthesis and degradation of glycogen in cultured rat myotubes. J Biol Chem 277: 4831–4838, 2002.[Abstract/Free Full Text]
  12. Frosig C, Jorgensen SB, Hardie DG, Richter EA, and Wojtaszewski JF. 5'-AMP-activated protein kinase activity and protein expression are regulated by endurance training in human skeletal muscle. Am J Physiol Endocrinol Metab 286: E411–E417, 2004.[Abstract/Free Full Text]
  13. Hansen BF, Derave W, Jensen P, and Richter EA. No limiting role for glycogenin in determining maximal attainable glycogen levels in rat skeletal muscle. Am J Physiol Endocrinol Metab 278: E398–E404, 2000.[Abstract/Free Full Text]
  14. Holmes BF, Kurth-Kraczek EJ, and Winder WW. Chronic activation of 5'-AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle. J Appl Physiol 87: 1990–1995, 1999.[Abstract/Free Full Text]
  15. Jansson E. Acid soluble and insoluble glycogen in human skeletal muscle. Acta Physiol Scand 113: 337–340, 1981.[ISI][Medline]
  16. Kiens B, Roepstorff C, Glatz JF, Bonen A, Schjerling P, Knudsen J, and Nielsen JN. Lipid-binding proteins and lipoprotein lipase activity in human skeletal muscle: influence of physical activity and gender. J Appl Physiol 97: 1209–1218, 2004.[Abstract/Free Full Text]
  17. Kraniou Y, Cameron-Smith D, Misso M, Collier G, and Hargreaves M. Effects of exercise on GLUT-4 and glycogenin gene expression in human skeletal muscle. J Appl Physiol 88: 794–796, 2000.[Abstract/Free Full Text]
  18. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970.[ISI][Medline]
  19. Lin A, Mu J, Yang J, and Roach PJ. Self-glucosylation of glycogenin, the initiator of glycogen biosynthesis, involves an inter-subunit reaction. Arch Biochem Biophys 363: 163–170, 1999.[CrossRef][ISI][Medline]
  20. Marchand I. A Quantitative Analysis of the Subcellular Distribution of Human Skeletal Muscle Glycogen (PhD thesis). University of Guelph: Guelph, ON, Canada, 2001.
  21. Marchand I, Chorneyko K, Tarnopolsky M, Hamilton S, Shearer J, Potvin J, and Graham TE. Quantification of subcellular glycogen in resting human muscle: granule size, number, and location. J Appl Physiol 93: 1598–1607, 2002.[Abstract/Free Full Text]
  22. Melendez R, Melendez-Hevia E, and Cascante M. How did glycogen structure evolve to satisfy the requirement for rapid mobilization of glucose? A problem of physical constraints in structure building. J Mol Evol 45: 446–455, 1997.[ISI][Medline]
  23. Melendez R, Melendez-Hevia E, Mas F, Mach J, and Cascante M. Physical constraints in the synthesis of glycogen that influence its structural homogeneity: a two-dimensional approach. Biophys J 75: 106–114, 1998.[Abstract/Free Full Text]
  24. Nielsen JN, Derave W, Kristiansen S, Ralston E, Ploug T, and Richter EA. Glycogen synthase localization and activity in rat skeletal muscle is strongly dependent on glycogen content. J Physiol 531: 757–769, 2001.[Abstract/Free Full Text]
  25. Pilegaard H, Keller C, Steensberg A, Helge JW, Pedersen BK, Saltin B, and Neufer PD. Influence of pre-exercise muscle glycogen content on exercise-induced transcriptional regulation of metabolic genes. J Physiol 541: 261–271, 2002.[Abstract/Free Full Text]
  26. Pilegaard H, Ordway GA, Saltin B, and Neufer PD. Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise. Am J Physiol Endocrinol Metab 279: E806–E814, 2000.[Abstract/Free Full Text]
  27. Pilegaard H, Saltin B, and Neufer PD. Exercise induces transient transcriptional activation of the PGC-1alpha gene in human skeletal muscle. J Physiol 546: 851–858, 2003.[Abstract/Free Full Text]
  28. Roach PJ. Glycogen and its metabolism. Curr Mol Med 2: 101–120, 2002.[Medline]
  29. Shearer J, Marchand I, Sathasivam P, Tarnopolsky MA, and Graham TE. Glycogenin activity in human skeletal muscle is proportional to muscle glycogen concentration. Am J Physiol Endocrinol Metab 278: E177–E180, 2000.[Abstract/Free Full Text]
  30. Smythe C, Watt P, and Cohen P. Further studies on the role of glycogenin in glycogen biosynthesis. Eur J Biochem 189: 199–204, 1990.[Abstract]
  31. Van Maanen MH, Fournier PA, Palmer TN, and Abraham LJ. Characterization of the human glycogenin-1 gene: identification of a muscle-specific regulatory domain. Gene 234: 217–226, 1999.[CrossRef][ISI][Medline]
  32. Wojtaszewski JF, MacDonald C, Nielsen JN, Hellsten Y, Hardie DG, Kemp BE, Kiens B, and Richter EA. Regulation of 5'-AMP-activated protein kinase activity and substrate utilization in exercising human skeletal muscle. Am J Physiol Endocrinol Metab 284: E813–E822, 2003.[Abstract/Free Full Text]
  33. Zong H, Ren JM, Young LH, Pypaert M, Mu J, Birnbaum MJ, and Shulman GI. AMP kinase is required for mitochondrial biogenesis in skeletal muscle in response to chronic energy deprivation. Proc Natl Acad Sci USA 99: 15983–15987, 2002.[Abstract/Free Full Text]