1 Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph N1G 2W1; and 2 Department of Medicine and Kinesiology, McMaster University. Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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The de novo biosynthesis of
glycogen is catalyzed by glycogenin, a self-glucosylating protein
primer. To date, the role of glycogenin in regulating glycogen
metabolism and the attainment of maximal glycogen levels in skeletal
muscle are unknown. We measured glycogenin activity after enzymatic
removal of glucose by -amylase, an indirect measure of glycogenin
amount. Seven male subjects performed an exercise and dietary protocol
that resulted in one high-carbohydrate leg (HL) and one
low-carbohydrate leg (LL) before testing. Resting muscle biopsies were
obtained and analyzed for total glycogen, proglycogen (PG),
macroglycogen (MG), and glycogenin activity. Results showed differences
(P < 0.05) between HL and LL for total glycogen (438.0 ± 69.5 vs. 305.7 ± 57.4 mmol glucosyl units/kg dry wt) and PG
(311.4 ± 38.1 vs. 227.3 ± 33.1 mmol glucosyl units/kg dry wt). A
positive correlation between total muscle glycogen content and
glycogenin activity (r = 0.84, P < 0.001) was
observed. Similar positive correlations (P < 0.05) were also
evident between both PG and MG concentration and glycogenin activity
(PG, r = 0.82; MG, r = 0.84). It can be concluded that
glycogenin does display activity in human skeletal muscle and is
proportional to glycogen concentration. Thus it must be considered as a
potential regulator of glycogen synthesis in human skeletal muscle.
glycogen metabolism; proglycogen; macroglycogen; carbohydrate loading; carbohydrate synthesis
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INTRODUCTION |
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GLYCOGEN SYNTHESIS IS CATALYZED by a self-glucosylating protein primer, glycogenin. Glycogenin autocatalytically generates an oligosaccharide primer of 7-11 glucosyl units, which serves as a substrate for glycogen synthase. Glycogen synthase and branching enzyme then act to catalyze the formation of two physiologically distinct pools of glycogen, pro- (PG) and macroglycogen (MG). PG and MG have identical protein contents but differ in the amount of associated carbohydrate (9). PG is a smaller glycogen entity, which has a molecular mass of up to 400,000 Da, whereas MG can reach a molecular mass of 107 kDa. Recent studies have shown PG and MG to be metabolically distinct as they differ in their rates of degradation and synthesis and in their sensitivities to dietary manipulation (1, 2).
Although the mechanisms controlling glycogen biogenesis are poorly understood, it has been suggested that glycogenin is a regulator of glycogen metabolism (8, 11, 12). In rabbit skeletal muscle, glycogenin is thought to be present in proportion to glycogen concentration (12). This has raised the possibility that glycogen storage is limited by the presence of the glycogenin (12). Glycogenin is also known to be complexed with glycogen synthase in skeletal muscle, and the relationship between the two enzymes may be important in controlling the rate of glycogen synthesis. Under resting conditions, no free deglucosylated glycogenin can be detected in skeletal muscle, indicating that all glycogenin is bound to glycogen (12). This may mean that there are no free stores of the protein available for the synthesis of additional glycogen molecules, and glycogenin may have to be synthesized as needed. This may explain why glycogen supercompensation under normal conditions can take days to complete.
Studies in rodent skeletal muscle have found the glycogenin protein to be active and to vary with fiber type but not with training status (6). Slow-twitch red fibers have the greatest glycogenin activity, followed by fast-twitch red and fast-twitch white muscle, which had the lowest activity. Glycogenin protein content also followed this order. Endurance exercise training resulted in no change in either glycogen activity or expression, and it was concluded that glycogenin did not play a role in limiting glycogen concentration in rodent skeletal muscle (6).
The only report of glycogenin activity on human skeletal muscle was
performed by Jiao et al. (7), who found no free glycogenin in human
skeletal muscle despite glycogen-depleting exercise. In this study, we
quantify glycogenin activity present in muscle after removal of
carbohydrate by -amylase. This was done over a range of
glycogen concentrations by measuring its ability to glucosylate an
exogenous substrate. This measure is an indirect measure of glycogenin amount.
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METHODOLOGY |
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Subject Characteristics
Seven male subjects completed the study. Their mean (± SE) age was 25.4 ± 1.1 yr; height, 182.6 ± 3.3 cm; weight, 89.7 ± 2.4 kg; one-leg maximal O2 uptake (Pretrial Procedures
Before the experiment, subjects were familiarized with the equipment and performed incremental one- and two-leggedGlycogen loading phase.
In an effort to lower muscle glycogen before carbohydrate loading,
subjects performed a two-legged exercise protocol on a cycle ergometer
(Lode, Netherlands). This was designed to deplete glycogen stores in
all fiber types. Exercise consisted of 1.5 h of cycling at 70%
O2 max
followed by a 10-min rest period. Subjects then performed five bouts of
3 min in duration at 90%
O2 max, with 1 min of
rest between bouts. Finally, after 5 min of rest, subjects completed 5 × 30 s of sprinting at maximal capacity or until exhaustion was
achieved in each bout. After the exercise protocol, subjects consumed a
carbohydrate supplement (Gatorlode) and a high-carbohydrate
snack before leaving the laboratory. A carbohydrate-loading protocol
was maintained for 2 days until the glycogen depletion phase was
initiated. Subjects maintained dietary records throughout the protocol.
Selective glycogen depletion.
Subjects depleted glycogen levels in one leg only on a modified cycle
ergometer (Monark). The one-leg depletion protocol consisted of
one-legged exercise for 1.5 h at 70% one-leg
O2 max, followed by
five bouts of 1 min in duration at 100% one-leg
O2 max. Subjects then
completed five maximal 30-s sprints. Rest periods between bouts were
the same as in the glycogen loading phase. Four subjects exercised
their dominant leg, while three exercised their nondominant leg. After
the exercise protocol, subjects maintained a low-carbohydrate diet for
36 h until the experiment was conducted. Dietary diaries for the high-
and low-carbohydrate diets were recorded.
Experimental Protocol
Subjects arrived at the laboratory after consuming a low-carbohydrate breakfastAnalyses
Glycogenin activity.
Glycogenin activity was measured as previously described (4, 6). The
term glycogenin activity refers to the ability of glycogenin to
transglucosylate a maltose derivative. This is an indirect measure of
glycogenin concentration. Wet muscle was ground with a mortar and
pestle under liquid nitrogen and weighed into 30- and 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 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 of -amylase (Sigma) for 1 h at 37°C.
The amylolysed sample was incubated in a mixture containing 8 µM
UDP-[14C]glucose (UPDG; ARC, 300 mCi/mmol), 17 mM MES (pH 7), 5 mM MnSO4, 0.2 mM
n-dodecyl-ß-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), UDPG (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 (Waters).
The cartridges were washed with 16 ml water, and the glucosylated
[14C]DBM eluted with 3 × 1-ml volumes 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 · min
1 · mg
protein
1 (4). The 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).
PG and MG. The remaining muscle samples were freeze-dried and dissected free of visible blood, connective tissue, and other nonmuscle elements. A 2- to 3-mg portion of freeze-dried muscle was extracted for enzymatic measurement of PG and MG as previously described (1). Glycogen values are reported in millimole glucosyl units/kg dry wt. Total glycogen is the sum of the PG and MG values.
Statistical Analysis
Data are presented as means ± SE. A paired t-test was used to compare differences between the HL and LL legs. Results are considered significant at P < 0.05. Relationships between glycogenin activity and PG, MG, and total glycogen were performed by a linear regression analysis. ![]() |
RESULTS |
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Glycogenin Activity
Human skeletal muscle glycogenin activity was found to range between 81.5 and 217.0 mU · mg protein
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PG and MG
PG and MG values in HL and LL are shown in Table 1. Significant differences (P < 0.05) between HL and LL were found for PG and total glycogen. Correlations between PG, MG, and glycogenin activity were also significant (P < 0.05; r = 0.82, PG; r = 0.85, MG; Fig. 2). The slopes of regression correlating glycogenin activity with PG and MG were 2.7 and 2.1, respectively.
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DISCUSSION |
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To date, little is known about the role of glycogenin in regulating glycogen metabolism. The primary purpose of this study was to establish the nature of glycogenin activity in human skeletal muscle and its relationship to glycogen concentration. This was done by quantifying glycogenin activity over a range of glycogen concentrations. Theoretically, the number of glycogenin molecules available within skeletal muscle would dictate the number of glycogen particles and the amount of glycogen stored. This suggests that glycogenin could be a factor in determining glycogen concentration. The present study supports this theory by showing a correlation between glycogenin activity and total glycogen concentration. This finding also supports the concept that glycogenin is present in proportion to glycogen concentration and that no free deglucosylated glycogenin exists in resting skeletal muscle.
Regression analysis showed comparable correlations between glycogenin activity and PG and MG, indicating no difference in glycogenin activity between the two pools of glycogen. It should be noted that, although glycogenin activity was not measured directly in each pool of glycogen, such a relationship would be expected, because the two molecules differ only in the amount of associated carbohydrate and not the amount of protein. Recent work by Hansen (6) with PG and MG has suggested that a major limiting step in glycogen synthesis is not glycogenin, but rather the transition from PG to MG. The rationale is that glycogen is rarely degraded to free glycogenin, and additional glucose molecules are added to existing moieties. In this case, a small amount of glycogenin would exist, and it would all be bound to glycogen. However, in examining the resynthesis of PG and MG, increases in PG are seen before increases in the MG pool (2). This would suggest that the additional glycogen is being synthesized on new glycogenin molecules rather than being added to existing ones. Electron micrographs also support this hypothesis, because new glycogen granules appear in skeletal muscle with carbohydrate loading and disappear with exercise (5).
The only other report examining glycogenin in human skeletal muscle was performed by Jiao et al. (7). They were able to show that MnSO4-dependent glycogen synthase was the major factor in the transfer of UDP-glucose to glycoprotein. The study also measured free glycogenin activity at rest and after exercise to fatigue. Results showed that the glycogenin protein was detectable by SDS-PAGE immunoblots; however, no free glycogenin activity could be detected, indicating that all of the protein was bound to carbohydrate. In the present study, glycogenin was deglycosylated to measure glycogenin through its ability to transglycosylate an exogenous substrate. We have shown the method employed in the present study to be internally consistent and proportional to glycogen amount.
Glycogenin has the potential to regulate glycogen metabolism at a number of sites. Hansen (6) has reviewed three possible sites of regulation on the protein itself. These include regulation by interacting with other proteins involved in glycogen metabolism, namely glycogen synthase; a phosphorylation site that would alter activity of the enzyme; and regulation by glucose. Under normal resting conditions, glycogenin is complexed to glycogen synthase. Together, these two proteins may interact to regulate glycogen synthesis. These two proteins are known to separate when skeletal muscle is exposed to prolonged electrical stimulation (12), or when muscle is treated with epinephrine (12). Both situations result in glycogen-free glycogenin not bound to glycogen synthase. Once dissociated, the reassociation of glycogenin to glycogen synthase is time dependent (12). This reassociation of glycogenin to glycogen synthase could be a point of regulating glycogen synthesis, which may link glycogen metabolism to hormonal and neural factors. Regulation by glucose or glucose-related proteins is another possibility. The interaction of glycogenin with GLUT-4 and the mechanism by which glucose is transferred to glycogenin have yet to be explored. Finally, glycogenin could be regulated by phosphorylation, as covalently bound phosphate is present in isolated skeletal muscle glycogenin extracts at 0.8 mol/mol (10a). A potential regulatory role for phosphate on glycogenin is unknown.
Another possible point of glycogenin regulation may be at the transcriptional or translational level. If no free deglucosylated glycogenin exists within the muscle, then it is assumed that it is undetectable by current methods, synthesized as needed by the muscle, or recycled. Recycling the protein would mean that the protein is somehow cleaved from the mature glycogen molecule and released to start resynthesizing more glycogen (11). In the present study, glycogenin activity increased in proportion to glycogen concentration. This suggests that the glycogenin protein is not recycled but rather synthesized as needed by the muscle. This may be the reason it takes days to resynthesize glycogen after a prolonged exercise bout such as a marathon (3). Asp et al. (3) measured PG and MG premarathon, postmarathon, and on days 1, 2, and 7 postmarathon. They showed that in the first couple of days after the marathon, PG and MG are rapidly resynthesized at similar rates. After 2 days of recovery, glycogen resynthesis continued in both pools at a lesser rate. This finding supports the hypothesis that glycogen is resynthesized on new glycogenin molecules and not added to existing glycogen structures, because both pools of glycogen increased at the same rate in the days after the marathon. If glucosyl units were added to existing structures, then one would expect a decrease in PG concentration with increasing MG concentration. Besides regulating glycogen resynthesis postexercise, the regulation of glycogenin may also be important in disease states such as type II (non-insulin-dependent) diabetes, where there is a marked impairment of glycogen synthesis.
In conclusion, this study has shown that glycogenin activity is correlated to glycogen concentration and is quantifiable in human skeletal muscle. Significant correlations exist for total glycogen as well as for PG and MG. Although the role of glycogenin in glycogen metabolism is unknown, it has the potential to be an important regulator of glycogen metabolism.
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ACKNOWLEDGEMENTS |
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The technical assistance of Bo Flack Hansen and Pia Jensen was much appreciated.
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FOOTNOTES |
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This study was supported by The Natural Sciences and Engineering Research Council of Canada (NSERC) and Gatorade Sport Science Institute. J. Shearer is supported by an Industrial NSERC award sponsored by Gatorade Sport Science Institute.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Shearer, Human Biology and Nutritional Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1 (E-mail: jshearer{at}uoguelph.ca).
Received 10 May 1999; accepted in final form 20 September 1999.
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