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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, Copenhagen, Denmark
Submitted 8 March 2005 ; accepted in final form 28 April 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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0.05). Exh resulted in decreases in total glycogen, proglycogen, and macroglycogen as well as glycogenin activity (P < 0.05). These decrements were associated with a 1.9 ± 0.4-fold increase in glycogenin mRNA over resting values (P < 0.05). Glycogenolysis in the initial exercise period (0–30 min) was not adequate to induce changes in glycogenin; however, later in exercise when concentration and granule number decreased further, decrements in glycogenin activity and increases in glycogenin mRNA were demonstrated. Results show that glycogenin becomes inactivated with glycogen catabolism and that this event coincides with an increase in glycogenin gene expression as exercise and glycogenolysis progress. glycogen granule; metabolism; glycogenolysis
Glycogenin initiates granule formation by the addition of 7–11 glucose residues to a single tyrosine residue on the protein. To date, two types of glycogenin have been identified: glycogenin-1 (GN-1) and glycogenin-2 (GN-2) (19). GN-1 (42 kDa) content is greatest in skeletal muscle with small amounts located in the liver, whereas GN-2 (66 kDa) is abundant in the heart and liver, with small amounts in the pancreas. The present study deals exclusively with GN-1 in skeletal muscle. GN-1 is unique in that it is all complexed to glycogen, and no free, deglycosylated protein has been detectable in skeletal muscle (29). This lack of available protein may be a point of regulation in glycogen synthesis, influencing the timing, location, extent, and type of glycogen granules formed.
Several reports have shown GN-1 mRNA to increase by approximately twofold following exercise (3, 16, 28). However, the relationship between GN-1 mRNA and protein activity, a measure of protein content, remains to be established. It is also unclear whether these changes are initiated during exercise itself in the presence of ongoing glycogenolysis or solely during glycogen resynthesis in the postexercise period. Whether a threshold decline in glycogen and/or granule number is required for the initiation of GN-1 mRNA transcription is uncertain, although previous studies have demonstrated PG to be a better predictor of GN-1 activity compared with MG (27).
To explore the effects of glycogenolysis on GN-1, the present study was performed. The fate of GN-1 during glycogen catabolism was determined by employing measures of PG and MG, GN-1 mRNA and activity during exercise. GN-1 activity is the ability of the protein to glycosylate and is an indirect measure of protein concentration. It was hypothesized that GN-1 activity would decrease during glycogen catabolism and that this would coincide with the disappearance of PG, the more dynamic form of glycogen.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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O2 max) 56.8 ± 3 ml·kg–1·min–1. The study received approval from the Human Ethics Committee at the University of Guelph. Subjects were informed of potential risks involved with the procedure, and consent was obtained. Participants were active, exercising two to three times per week, but refrained from exercise 3 days before the experimental protocol.
Pretrial procedures.
Two weeks before the experimental protocol, subjects performed an incremental exercise test to determine O2 max. One week before the experiment, subjects returned to the laboratory to be familiarized and perform a 45-min practice ride at 75% of their O2 max. Three days before the experimental protocol, subjects consumed their normal diet but refrained from caffeine, alcohol, and aspirin.
Experimental protocol.
On the day of the experiment, subjects consumed a high-carbohydrate breakfast (
70% of calories derived from carbohydrate) at least 2 h before the exercise trial. A catheter was placed in the medial anticubital vein for blood sampling, and a resting muscle biopsy was obtained (0 min) from the vastus lateralis by using a percutaneous needle biopsy technique under local anesthesia. Subjects then exercised at 75% of their O2 max on a cycle ergometer. Another biopsy was obtained at 30 min. Following this, subjects continued to exercise to volitional exhaustion (Exh) at which time a final muscle biopsy was obtained. During the ride, subjects were provided with water at regular intervals. Blood samples (5 ml) for the measurement of plasma glucose and insulin were taken at 0 min, 30 min, and Exh.
Analyses. Muscle biopsies were rapidly frozen in liquid nitrogen and stored at –80°C until further analysis.
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 2–3 mg dry wt (dw) portion were analyzed enzymatically, as previously described, and are reported in millimoles glucosyl units per kilogram dw (1, 2, 17). Briefly, ice-cooled 1.5 M perchloric acid (PCA) (200 µl) were added to 1.5–3 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 of the muscle was exposed to acid. The extraction continued on ice for 20 min. The samples were centrifuged at 3,000 revolutions/min 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 revolutions/min for 5 min, and transferred to Eppendorf tubes for analysis of glucosyl units by using the method of Bergmeyer (5) or stored at –80°C. Total glycogen (Gt) is the sum of the PG and MG values.
RNA isolation. Total RNA was extracted from 30 mg of muscle by a modified Chomczynski and Sacchi method using TRIzol reagent (GIBCO-BRL) (4). 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 2 x 0.5 ml ethanol, air-dried, and then resuspended in 20 µl of RNase-free water. Concentration and purity of the RNA isolation were determined on 1 µl of the extract by spectroscopy.
RT. RT of samples was performed using the Thermoscript RT-PCR system (GICBO-BRL). Oligo(dT20) primers were combined with 1 µg of total RNA and diethyl pyrocarbonate-treated water (to 10 µl). RNA was denatured by heating for 5 min at 65°C before the 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 units RnaseOUT (GIBCO-BRL), 1 mM dNTP mix, 1.5 units RT (Thermoscript, GICBO-BRL), and 1 µl of diethyl pyrocarbonate water. Samples were gently mixed and then transferred to a thermocycler (Techgene, Cambridge), where samples were heated to 55°C for 45 min and then to 85°C for 5 min to terminate the RT reaction. RNase H (GICBO-BRL) was added, and samples were incubated at 37°C for 20 min before being stored at –20°C until further analysis.
PCR.
Before PCR of experimental samples, optimal, nonsaturating conditions for PCR were established (annealing temperature, number of cycles, MgCl2 concentration). All samples from a given subject were run simultaneously. mRNA content was determined in duplicate by PCR. The PCR reaction mixture was 50 µl [RT, 20 mM Tris·HCl (pH 8.4); 50 mM KCl; 0.2 mM each of dCTP, dATP, dGTP, dTTP; 1.5 mM MgCl2; 0.5 mM each of forward and reverse primers; and 1.25 units Taq DNA polymerase (GIBCO-BRL)]. PCR products were separated on 2.5% agarose gel 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, NIH) was then used to quantify the PCR products. To correct for differences in mRNA qualities, 
-actin mRNA was also amplified and used as a control in each PCR reaction. Forward and reverse -actin primers were 5'-CCCAAGGCCAACCGCGAGAGAT-3' and 5'-GTCCCGGCCAGCCAGGTCCAG-3', respectively, and resulted in a 219-bp product. The PCR cycle profile for -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, and 72°C for 5 min. GN-1 mRNA was quantified by 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, and 72°C for 5 min. To confirm that the expected genes were amplified, the gel-purified (GIBCO-BRL Concert Nucleic Acid Purification System) PCR products for both -actin and GN-1 were sequenced by an ABI automated (ABI Prism 377) sequencer at the University of Guelph Molecular Supercenter.
Glycogenin activity.
The term GN-1 activity refers to the ability of GN-1 to transglucosylate a maltose derivative. Specifically, it is the maximal ability of the protein to glycosylate under optimal conditions. As such, activity measures are an indirect quantification of the amount of protein present. GN-1 activity was measured as previously described (12, 13, 27). Briefly, 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 phenylmethylsulphonyl 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 was retained, and the myofibrillar pellet discarded. Protein concentrations were measured (Pierce, Coomassie Plus Protein Reagent Kit). Equal amounts of protein (150 µg) were amylolysed with 10 µg/ml of 
-amylase (Sigma) for 1 h at 37°C. The amylolysed sample was incubated in a mixture containing 8 µM UDP [14C]glucose (ARC, 300 mCi/mmol), 17 mM MES (pH 7), 5 mM MnSO4, 0.2 mM n-dodecyl -D-maltoside (DBM) (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), UDP-glucose (20 µl, 20 mM), and deionized water (Milli-Q, 84 µl) 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 through prewashed C18 Sep-Pak cartridges. The cartridges were washed with 16 ml of water, and the [14C]glucosylated DBM was eluted with 3 x 1 ml of methanol. Scintillation fluid was added (10 ml), 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 milligram 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).
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, and serum was then extracted and stored for the measurement of serum insulin. Insulin measurements were determined by using the radioimmunoassay method (RIA, Coat-a-Count, Diagnostic Products). The second aliquot (100 µl) of whole blood was added to 500 µl of 0.6 M PCA and centrifuged, and the supernatant was stored at –20°C for glucose analysis (5).
Calculations and statistical analysis. To calculate the percentage change in PG, MG, and Gt, values are expressed relative to Gt and not the resting values for each respective form of glycogen. For example, the percentage of PG catabolized at 30 min and Exh is expressed as (PG catabolized)/(Gt). Absolute values have been included beside each stated percentage for clarification. Data are presented as means ± SE. A one-way repeated-measures ANOVA test was used to establish differences in GN-1 activity, GN-1 expression, and PG, MG, and Gt between time points. Significant differences were located by a Tukey post hoc test. Differences were considered significant at P < 0.05. Relationships between GN-1 activity and PG, MG, and Gt were performed by a linear regression analysis.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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34% of glycogen remained in skeletal muscle at Exh. This inability to drive glycogen values lower may have been due to the training status of the athletes who were considered "recreationally active," exercising two to three times per week.
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-actin mRNA.
Measurements of both -actin and GN-1 mRNA were compared with the resting levels that were arbitrarily set to a value of 1. No change in GN-1 mRNA was observed at 30 min; however, at Exh, levels were significantly increased over resting levels. Values for GN-1 mRNA were 1.0 ± 0.0, 1.33 ± 0.14, and 1.94 ± 0.36 arbitrary units for 0 min, 30 min, and Exh, respectively (Fig. 2). Sequencing of GN-1 and -actin bands obtained from gels confirmed that they were expected products (data not shown). Blood glucose and serum insulin. Blood glucose steadily declined from 4.44 ± 0.27 mM at rest to 3.60 ± 0.34 mM at 30 min and 3.07 ± 0.31 mM at Exh. Plasma insulin followed a similar trend with values of 29.77 ± 10.7, 6.22 ± 2.59, and 1.93 ± 0.73 µU/ml, respectively.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The exercise protocol in the present study was divided into two time intervals: rest to 30 min and 30 min to Exh. In the initial period, there were no significant changes in glycogen concentration, and it is likely that few granules were totally degraded. This may explain the lack of change in GN-1 activity and mRNA during this period. In contrast, glycogenolysis was associated with significant changes in GN-1 activity and mRNA in the second period. Such changes likely occurred due to complete degradation of individual granules. This finding is supported by Elsner et al. (8), who have demonstrated that the number of glycogen granules in the initial phases of glycogenolysis remains constant, while subsequent time intervals are characterized by the complete degradation of individual glycogen granules in cultured rat myotubes. Thus the greater declines in GN-1 activity from 30 min to Exh may, in part, be due to the marked catabolism of glycogen granules, although a time effect of exercise itself cannot be excluded. It is evident from this and previous studies examining glycogenin activity that there is a strong correlation between this variable and glycogen concentration. In the present study, glycogenin activity declined as glycogenolysis proceeded. In contrast, a study by Shearer et al. (28) shows that, during glycogen resynthesis following exercise, glycogenin activity levels increase. Together, these results indicate an apparent codependence of glycogenin activity on glycogenin concentration in human skeletal muscle.
Declines in GN-1 activity with exercise in the present study likely signify either inactivation of the protein or its degradation. GN-1 catalyzes the addition of a single UDP-glucose by a C-1-O tyrosyl linkage at amino acid residue Tyr194. Mutation studies have shown that altering this site to either phenylalanine or threonine renders GN-1 inactive, showing that this residue is essential for self-glycosylation (6). Following this initial glucosyl addition to Tyr194, GN-1 adds additional UDP-glucose residues to form an oligosaccharide chain of 7–11 glucosyl units in length, each attached by -1,4 linkages. Once complete, this chain is acted upon by glycogen synthase and phosphorylase. In the case of degradation by glycogen phosphorylase, it is possible that all residues are removed except for the last one attached by Tyr194, whose bond (C-1-O tyrosyl linkage) is not accessible by the enzyme. If this is the case, then it may be that this bond renders the protein inactive in vivo and results in the dissociation of glycogen synthase. Such a mechanism would be sensible, as it would be disadvantageous to have GN-1 competing for available glucose residues during high rates of glycolysis. Given this, the end product of glycogenolysis would be GN-1 with a single glucose residue attached of 42 kDa in size. This transition in size explains previous findings of Nielsen et al. (20), who show translocation of glycogen synthase from a glycogen-enriched membrane fraction to a cytoskeletal fraction on glycogen depletion. Translocation likely reflects the GN-1 (plus attached glucose entity that would result) movement from a high-carbohydrate content (soluble) to a protein complex comprised mainly of protein (insoluble). Additional studies examining GN-1 protein content in relation to its activity in skeletal muscle during glycogenolysis are needed to confirm this hypothesis. Unfortunately, antibody for the quantification of GN-1 was not available at the time of this study.
We show declines in GN-1 activity to be accompanied by increases in GN-1 mRNA. Increases in GN-1 mRNA only occurred once glycogen concentrations declined in the second period. Specifically, it may be a fall in PG concentration and/or granule number that triggers GN-1 mRNA. Intuitively, this is sensible as declines in PG would result in a greater rate of granule liberation compared with MG. Similar increases in GN-1 mRNA have been shown to occur in the postexercise period (3, 16). Of particular interest is a study by Arkinstall et al. (3), who demonstrated that alterations in GN-1 mRNA were sensitive to diet. Specifically, individuals fed a high-carbohydrate diet following exercise had a 1.6-fold increase in GN-1 mRNA compared with counterparts who consumed a low-carbohydrate diet. The same magnitude of mRNA change was observed in the present study. Although GN-1 protein synthesis cannot be inferred from the present results or those of Arkinstall et al., the results strongly suggest that at least a portion of the transcript was translated into functional protein to facilitate GN-1 resynthesis. While the increases in mRNA were not large, it is important to note that a small amount of protein could store a large amount of carbohydrate.
It is likely a factor associated with exercise, or even glycogen depletion itself, that triggers transcription of GN-1. Such a transcription factor could also be integral to triggering other metabolic genes in the exercise response. Indeed, Pilegaard and coworkers (9, 14, 15, 21–26) have demonstrated that exercise and glycogen concentration are powerful regulators of gene expression of interleukin-6, pyruvate dehydrogenase kinase 4, uncoupling protein 3, lipoprotein lipase, and hexokinase II mRNA. Due to the similar time course of gene upregulation with exercise, it has been suggested that a common transcription factor is responsible. Possible regulators may include peroxisome proliferator activated receptor coactivator-1, a nuclear factor that increases up to 40-fold in response to exercise, or AMP kinase, a kinase activated in response to fluctuations in the energy status of the myocyte (7, 26, 30, 31).
In summary, we show GN-1 activity to decline with exercise, suggesting that the protein is degraded or inactivated. This inactivation, although wasteful, may negate competition for free glucose residues between GN-1 and ongoing glycolysis during exercise. Glycogenolysis or exercise itself also trigger GN-1 mRNA expression, an event that may be essential to glycogen resynthesis in the postexercise period.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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