1 Diabetes Biology, Novo Nordisk, DK-2880 Bagsværd; and 2 Copenhagen Muscle Research Centre, August Krogh Institute, University of Copenhagen, DK-2100 Copenhagen, Denmark
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ABSTRACT |
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We examined whether the protein level and/or activity of glycogenin, the protein core upon which glycogen is synthesized, is limiting for maximal attainable glycogen levels in rat skeletal muscle. Glycogenin activity was 27.5 ± 1.4, 34.7 ± 1.7, and 39.7 ± 1.3 mU/mg protein in white gastrocnemius, red gastrocnemius, and soleus muscles, respectively. A similar fiber type dependency of glycogenin protein levels was seen. Neither glycogenin protein level nor the activity of glycogenin correlated with previously determined maximal attainable glycogen levels, which were 69.3 ± 5.8, 137.4 ± 10.1, and 80.0 ± 5.4 µmol/g wet wt in white gastrocnemius, red gastrocnemius, and soleus muscles, respectively. In additional experiments, rats were exercise trained by swimming, which resulted in a significant increase in the maximal attainable glycogen levels in soleus muscles (~25%). This increase in maximal glycogen levels was not accompanied by an increase in glycogenin protein level or activity. Furthermore, even in the presence of very high glycogen levels (~170 µmol/g wet wt), ~30% of the total glycogen pool continued to be present as unsaturated glycogen molecules (proglycogen). Therefore, it is concluded that glycogenin plays no limiting role for maximal attainable glycogen levels in rat skeletal muscle.
fiber type; training; supercompensation; proglycogen; macroglycogen
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INTRODUCTION |
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THE GLYCOGEN PROTEIN CORE was named glycogenin in 1985 (14) and was identified as the minor 37-kDa subunit of glycogen synthase existing in a 1:1 molar ratio with the catalytic 86-kDa subunit (20). Glycogenin and glycogen synthase have also been shown to coimmunoprecipitate from extracts of cultured cells by use of anti-glycogenin antibodies (27). Glycogenin was found not simply to act as a protein backbone but also to be an enzyme itself that catalyzed its own glucosylation, and it is classified as a hexosyltransferease (EC 2.4.1.186) (3, 16, 21, 32).
Theoretically, glycogen particles must have molecular weights between
that of glycogenin (37 kDa) and the maximal attainable size, depending
on how many glucose units it contains. The fully "mature"
glycogen -particle has a molecular weight of
~1 · 107, which means that the
molecular weight of glycogenin is 0.35% of the
-particle.
Interestingly, when the protein content of glycogen was measured, it
came to exactly that number: 0.35%, suggesting that glycogenin and
-particles exist in a 1:1 stoichiometric relationship (14, 28). Thus
it follows that there is an average of one glycogen synthase catalytic
subunit bound to each glycogen molecule (32). In skeletal muscle it was
observed that almost all of the glycogenin was bound covalently to the
carbohydrate moiety and that no reservoir of free glycogenin molecules
exist (17, 33). Therefore, the amount of glycogenin present in muscle could be the factor ultimately limiting glycogen deposition, because glycogen synthesis would stop if all glycogenin molecules were saturated (32).
Skurat et al. failed to observe an increase in total cell glycogen in COS (31) or rat 1 fibroblasts (30) overexpressing glycogenin. However, in these studies cells were only stimulated with glucose, not with insulin, and cells were only studied at a single time point. Therefore, in these studies it is unknown whether the maximal glycogen level was reached. In fact, a small but significant increase in maximal attainable glycogen level was observed in L6 cells overexpressing glycogenin (Hansen, unpublished observations). Nevertheless, neither of these studies allows any firm conclusions with respect to skeletal muscle, because major differences exist between cultured cells and skeletal muscle.
Thus the purpose of this study was to investigate whether maximal attainable glycogen levels in skeletal muscle are determined by the protein level and/or activity of glycogenin. For this purpose we studied glycogenin protein levels and activity in different fiber types and in soleus muscles after exercise training. Furthermore, we examined the proportion of pro- and macroglycogen in skeletal muscles from rats with very different levels of glycogen.
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MATERIALS AND METHODS |
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All experiments were approved by the Danish Animal Experiments Inspectorate and complied with the "European Convention for the Protection of Vertebrate Animals Used for Experiments and Other Scientific Purposes" (Council of Europe no. 123, Strasbourg, France, 1985).
The fiber type study.
Fed male Wistar rats (~210 g) from Møllegård (Lille
Skensved, Denmark) were anesthetized by a brief exposure to
CO2 and killed by cervical dislocation. The superficial
part of the white gastrocnemius muscle (WG), which consists mainly of
fast-twitch white (FTW) fibers (4), was cut off and freeze-clamped.
Then the soleus muscle (SOL), which consists mainly of slow-twitch red
(STR) fibers (4), was reflected and clamped. Finally, a
portion of the deep part of the medial head of the red gastrocnemius
(RG), consisting mainly of fast-twitch red (FTR) fibers (4), was cut
out and clamped. Muscles were stored at 80°C until analyzed.
Glycogen loading in soleus muscles.
In preparation for the training study, rat soleus muscles were
incubated in high glucose and insulin concentrations for 6 h to
determine the time course for saturation of glycogen stores. Fed male
Wistar rats (~50 g) from Møllegård were anesthetized by a
brief exposure to CO2 and killed by cervical dislocation. Soleus muscles were quickly removed (<1 min) and placed in 2 ml (31°C) of a pregassed (95% O2-5% CO2)
Krebs Henseleit (KRH) buffer (118.5 mM NaCl, 4.7 mM KCl, 1.2 mM
KH2PO4, 25 mM NaHCO3, 2.5 mM CaCl2, 1.2 mM MgSO4, 0.1% BSA, and 5 mM HEPES)
supplemented with 0.15 mM pyruvate, amino acids at concentrations
corresponding to DMEM (GIBCO), and 25 mM mannitol. The muscles were
preincubated for 30 min to equilibrate. At time 0 muscles were
changed to a new identical KRH buffer now containing high insulin (60 nM) and glucose (20 mM) concentrations, as well as 5 mM mannitol, and incubated for 0-6 h. Throughout the incubation, vials were gassed with 95% O2-5% CO2. The medium was replaced
with a fresh one every hour. At time 0 and 1, 2, 3, 4, 5, and 6 h, muscles were quickly freeze-clamped between tongs cooled in liquid
nitrogen. All muscles were kept at
80°C until analyzed.
The training study.
Male Wistar rats (50 g) were purchased from Panum Institute Breeding
Centre (Copenhagen University, Copenhagen, Denmark) and randomly
assigned to either a sedentary control group or a training group. The
rats were kept in cages (4 rats/cage) measuring 30 × 45 × 25 cm in a temperature (21°C)-controlled room with a 12:12-h light-dark cycle. The rats had free access to water and rat chow. The
rats assigned to the exercise training were acclimated to swimming by
swimming in water maintained at 35 ± 1°C for 10 min the 1st day,
1 h the 2nd day, and 3 h the 3rd day. From days 4-8 (5 days in total), the rats swam 2 times for 3 h separated by a 1-h rest
period per day. During the rest period rats were towel dried, kept
warm, and given food and water. The rats in both groups were weighed
before and after the training period. A similar training regimen has
previously been demonstrated to induce pronounced effects on glucose
metabolism in skeletal muscle (23, 24). After the last training
session, rats were allowed to rest for 40 h before experiments started.
The in vivo glycogen loading study. To examine the relationship between total glycogen and the proportion between pro- and macroglycogen, we subjected male Wistar rats (Panum Institute Breeding Centre) weighing 65-100 g (n = 17) to 2 h of swimming in water maintained at 32-35°C, with weights (6% of body wt) attached to their tails. In the 24 h preceding the swim, food intake of the rats was restricted to 4 g of rat chow. After swimming, they were fed ad libitum with either rat chow, tap water, and a 20% glucose drinking solution, or tap water and butcher's lard. At 18-24 h after the swimming bout, rats were anesthetized by an intraperitoneal injection of pentobarbital sodium (5 mg/100 g body wt). Muscle samples were taken for proglycogen and macroglycogen determination, as described in the fiber type study (WG, RG, and SOL). Additionally, the plantaris muscle was sampled. Rats were killed by an intracardial injection of pentobarbital sodium.
Glycogen content. Glycogen content was measured after acid hydrolysis by a hexokinase (HK) method (13). In short, 5- to 10-mg samples of muscle (wt/wt) were boiled in 1 ml HCl (1 N) for 2 h. Subsequently, ATP, HK, NADP+, and glucose-6-phosphate dehydrogenase were added, and the formation of NADPH was measured with a fluorometer at 455 nm.
Pro- and macroglycogen. Proglycogen and macroglycogen were separated as described by Adamo and Graham (1) by exposing biopsies to 1.5 M perchloric acid for 20 min on ice. Subsequently, the glycogen content in each fraction was measured as described above for glycogen.
Glycogenin protein level.
Frozen muscle biopsies were homogenized essentially as described (33).
In short, muscles were minced in a mortar in liquid nitrogen and
subsequently homogenized in 5 vol of buffer A [4 mM EDTA
(pH 7.0), 0.1 mM phenylmethylsulfonyl fluoride, 0.1% (by vol)
2-mercaptoethanol, and 1 mM benzamidine] at 4°C. The
homogenates were centrifuged (35 min, 4,200 g, 4°C), and
the myofibrillar pellet was discarded. Protein concentrations were
measured with the Bio-Rad Coomassie protein kit. Equal
amounts of protein (10 µg) were treated with -amylase (Sigma,
final concentration 10 µg/ml) for 1 h at 37°C. In some cases,
samples were also incubated for 1 h at 37°C without amylase.
Samples were subsequently analyzed by Western blotting. After SDS-PAGE
electrophoresis [10% Bis-Tris gels (Nu Page)], proteins
were transferred to polyvinylidene fluoride membranes, and the
membranes were blocked overnight in Tris-buffered saline (TBS) with 5%
BSA (Sigma) and 2% skim milk (DIFCO) at 4°C under gentle
agitation. To quantify glycogenin protein level, blocked membranes were
incubated with guinea pig anti-human glycogenin antibody (kindly
donated by Dr. Roach, Indiana University, Bloomington, IN) diluted
1:2,000 for 2 h at room temperature. After four washes in TBS with
0.05% Tween-20, membranes were incubated with goat anti-guinea pig IgG
horseradish peroxidase-conjugated antibody (Chemicon) diluted 1:3,000
for 1 h. Subsequently, separated proteins were visualized by enhanced
chemiluminescence (Amersham) according to the manufacturer's protocol.
His-tagged recombinant human glycogenin (kindly donated by Dr. Roach)
was used as a positive control. Bands were quantified by use of the
FujiFilm CCD camera and the Image Gauge software (Fuji Photo Film).
Glycogenin activity.
Glycogenin activity was measured by a modification of the method
described by Carizzo et al. (8). Muscle homogenates were prepared as
described above for glycogenin protein level. Equal amounts of protein
(150 µg) were subjected to amylolysis for 1 h at 37°C with
-amylase (Sigma, final concentration 10 µg/ml). Subsequently, the
activity of glycogenin was measured. The incubation mixture contained
the following components in a final volume of 60 µl: 8 µM
UDP-[14C]glucose (287 mCi/mmol, NEN), 17 mM MES
(pH 7.0), 5 mM MnSO4, 0.2 mM n-dodecyl
-D-maltoside (DBM; Sigma), and 50 µl
homogenate (150 µg protein). The glucosylation was allowed to proceed
for 10 min at 30°C, and the reaction was terminated by addition of 16 µl of 0.1 M EDTA. The samples were made 1 mM in glucose and 2 mM in cold UDP-glucose in a final volume of 200 µl.
Total radioactivity was measured in 10 of these 200-µl samples, and
the remainder were passed through a prewashed C18 cartridge
(Waters). The cartridges were washed with 10 ml of water, and the
14C-labeled glucosylated DBM was eluted with 3 times 1 ml
methanol. The three fractions per sample were counted (Packard,
TRI-CARB 1500) individually after addition of 10 ml scintillation
liquid (Ultima Gold). The majority (>95%) of the radioactivity was
recovered in the first two fractions, indicating that all glucosylated
DBM was eluted from the cartridges. One unit of activity is defined as
1 nmol of [14C]glucose incorporated into DBM
per minute (8).
Statistics. Statistical evaluation of the data was done by unpaired t-tests or one-way ANOVA by use of the Student-Newman-Keuls method for post hoc multiple comparisons, where appropriate. Data shown in Fig. 6 were fitted using the SigmaPlot for Windows version 4.0 (SSPS). Data are presented as means ± SE, and the level of significance was chosen at 0.05.
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RESULTS |
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Glycogenin activity was found to be significantly different in the
three fiber types (P < 0.001). The glycogenin activity was
highest in STR fibers, lowest in FTW fibers, and intermediate in FTR
fibers (Fig. 1). A quite similar fiber type
dependency of glycogenin protein levels was seen (ANOVA, P < 0.05, Fig. 1).
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Initial experiments showed that glycogen levels in SOL muscle could be
saturated within 5 h if incubated in a medium containing 20 mM glucose
and 60 nM insulin (data not shown). At the same time as glycogen stores
were saturated, glycogen synthesis rate decreased to 20% of the
initial value (Fig. 2). Therefore, SOL muscles from exercise-trained and untrained rats were incubated for 5 h
under these conditions (Fig. 3). In both
trained and untrained muscles, glycogen levels were found to be
saturated within 5 h, because no significant increase was seen from 4 to 5 h. Analysis of variance (ANOVA) revealed a significant overall
difference in glycogen between the two groups (P < 0.001).
Despite this increase in glycogen deposition in trained muscle, no
increase in glycogenin protein level or glycogenin activity could be
observed (Fig. 4).
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The concentrations of proglycogen and macroglycogen in the fed state in
the three different fiber types are shown in Fig. 5A. In all three fiber types, most
glycogen was found to be present as proglycogen. A fiber type
dependency of the proglycogen level was observed. Interestingly, the
proglycogen-to-macroglycogen ratio was found to be ~8 in all three
fiber types (Fig. 5B). The relative proportion of proglycogen
and macroglycogen was dependent on the total glycogen concentration
(Fig. 6). Thus, in muscles with a low total
glycogen concentration, almost all glycogen was present as proglycogen.
The relative proportion of proglycogen decreased from values close to
100% down to ~30% as the total glycogen concentration increased.
Interestingly, this relationship between the relative proportion of
proglycogen (or macroglycogen) and total glycogen seems to be
independent of fiber type (Fig. 6). Furthermore, it should be noted
that, despite a decrease in the relative proportion of proglycogen, an
increase in the absolute proglycogen concentration was in fact observed
as total glycogen increased.
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DISCUSSION |
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This study shows that the protein level and the activity of glycogenin are significantly different in the three major muscle fiber types (Fig. 1). Previously we showed that maximal glycogen levels were reached within 5 h in rat skeletal muscle when perfused with 11-13 mM glucose and insulin concentrations of maximal stimulation (25). The maximal attainable glycogen levels were found to be 137.4 ± 10.1, 80.0 ± 5.4, and 69.3 ± 5.8 µmol/g wet wt in FTR, STR, and FTW fibers, respectively (25). Thus there seems to be a poor correlation between maximal attainable glycogen levels during insulin stimulation and the protein level/activity of glycogenin in the three different fiber types.
Exercise training is normally associated with increased glycogen levels in muscle (12, 26). However, from these studies it is not possible to conclude that exercise training increases maximal attainable glycogen levels, because it is unknown whether maximal glycogen levels were in fact reached. Therefore, we incubated trained and untrained soleus muscles in high glucose and insulin concentrations for 5 h to saturate glycogen stores. Here we show that even short-term exercise training (5 days) does in fact increase maximal attainable glycogen levels (Fig. 3). Despite this increase in maximal glycogen levels, we failed to observe any increase in glycogenin protein level and activity. In fact, a small decrease in glycogenin activity was seen in trained muscle (Fig. 4). Thus, from the study of glycogenin protein level and activity in different rat muscle fiber types as well as in trained vs. untrained rat soleus muscle, it is suggested that glycogenin does not play a direct role for maximal attainable glycogen levels.
This conclusion is further supported by the study of relative proportions of proglycogen and macroglycogen in skeletal muscle. In the present study, an ~8:1 glycogen by weight proglycogen-to-macroglycogen (pro/macro) ratio was found in all three fiber types in the normal fed state (Fig. 5), which corresponds nicely to the values obtained by Adamo and Graham (1) in mixed human and rat muscle. The comparable pro/macro glycogen ratio found in the three fiber types, despite quite different levels of maximal attainable glycogen levels, indicates that glycogen synthesis stops before all proglycogen is converted to macroglycogen. This is further supported by the relationship between total glycogen and the relative proportions of proglycogen and macroglycogen (Fig. 6). Macroglycogen accounted for a minor portion of total glycogen at normal glycogen levels (~30 µmol/g wet wt), but the proportion of macroglycogen increased gradually. However, the proportion of macroglycogen never exceeded 70% of the total glycogen, even if total glycogen levels further increased from 100 to 170 µmol/g wet wt. So, even at very high glycogen levels, there is still up to 30% of the total glycogen that is in the proglycogen form, and the absolute proglycogen concentrations showed no sign of a decrease. Similar results were obtained by Adamo and colleagues (1, 2). In other words, even at very high glycogen concentrations, a considerable number of the glycogen particles are still unsaturated.
In the normal fed resting state, we found an ~8:1 glycogen by weight pro/macro ratio in all three fiber types. With the assumption of a molecular mass of 400,000 Da for proglycogen and 107 Da for macroglycogen (3), it can be calculated that, if all the remaining proglycogen were to be converted to macroglycogen, the glycogen levels would theoretically be increased ~20-fold to ~600 µmol/g wet wt. Interestingly, when the maximal glycogen concentration is estimated (when macroglycogen equals 100%) from the fit of the data on Fig. 6, a value of ~700 µmol/g wet wt is obtained. Therefore, we believe that the theoretical maximal glycogen concentration in rat skeletal muscle is ~600-700 µmol/g wet wt, which is three- to fourfold higher than the highest observed values (25). Thus, from the data in this study, it seems safe to conclude that glycogenin does not play a limiting role in determining maximal glycogen levels in rat skeletal muscle, because glycogen synthesis stops before all glycogen particles are saturated.
According to the model proposed by Alonso et al. (3), glycogen exists in various stages between proglycogen (400 kDa) and macroglycogen (107 Da). Furthermore, proglycogen is proposed to be a stable intermediate in the synthesis of macroglycogen, and it is never broken down to glycogenin (3). If it is assumed that the number of glycogenin molecules is constant, another interesting finding can be gained from this study, that the average size of proglycogen molecules increases with increasing total glycogen levels, because the absolute proglycogen concentration in fact increases. Thus the term proglycogen does not represent a distinct fraction (400 kDa) of the glycogen pool. If the theoretical maximal glycogen concentration is 700 µmol/g wet wt (corresponding to 12.6 nmol/g glycogenin), it can be calculated from the fit that, at normal fed glycogen levels (20-30 µmol/g wet wt), the average size of proglycogen molecules should be ~270 kDa, whereas the average size increases to ~575 kDa at a total glycogen of 90 µmol/g wet wt. At 650 µmol/g wet wt (just before the very last proglycogen molecules are converted into macroglycogen), the average size of proglycogen is 940 kDa, indicating that the critical size of proglycogen is 1,000 kDa, corresponding to a protein content of ~4%.
If glycogenin is not the factor ultimately limiting further synthesis of glycogen, despite ongoing stimulation with insulin and glucose, it must be assumed that some process during the storage of glycogen induces a state of insulin resistance responsible for the downregulation of glycogen synthesis. These processes could be partly responsible for the insulin resistance seen in type II diabetes. Alternatively, it could be speculated that glycogen breakdown was accelerated, which limited further increase in the glycogen levels despite the ongoing glycogen synthesis. The profound decrease in incorporation of [14C]glucose into glycogen in rat soleus muscles incubated in high glucose and insulin, as glycogen stores saturate, suggests that synthesis was in fact decreased (Fig. 2). Furthermore, glycogen synthase (GS) activity was found to be severely decreased in all three fiber types after 5 h of rat hindquarter perfusion with moderately high glucose (11-13 mM) and maximal stimulations of insulin (25). Therefore, we believe that prolonged exposure to high glucose and insulin induces a state of insulin resistance to glycogen synthesis in skeletal muscle.
Numerous studies have shown that, as glycogen stores increase after glycogen-lowering exercise, GS activity and glycogen synthesis rate decrease (5, 6, 9-11, 15, 19, 22, 29, 34). Furthermore, a significant negative correlation between GS activity and the glycogen concentration has been demonstrated (5, 6, 9, 10, 19, 34). This negative relationship between glycogen levels and glycogen synthesis was also found when glycogen levels were increased above normal values by stimulation with insulin and glucose (25; and Hansen, unpublished observations). Although these findings do not prove a direct role of glycogen in determining glycogen synthesis, they do suggest it. How such a mechanism might operate can only be speculated on at the present time, but it may be related to the binding of glycogen-metabolizing enzymes to the glycogen molecules. One hypothesis could be that the activity of GS is under the influence of the interaction with glycogenin. If GS activity is high only so long as glycogenin and GS are bound to each other, that circumstance could explain why GS activity decreases as the glycogen particle grows. Although there is no evidence for such a mechanism at the present time, it is intriguing that, in vitro, it was shown that GS only efficiently elongated the primer if complexed to glycogenin (33) and that the GS activity with respect to proglycogen and macroglycogen appears to be different (2, 18).
In summary, it was found in rat skeletal muscle that the protein level and activity of glycogenin correlated poorly with the maximal attainable glycogen level. In addition, a training-induced increase in maximal glycogen levels was not accompanied by an increase in either glycogenin protein levels or activity. Finally, even at very high glycogen levels, a considerable number of the glycogen particles are still unsaturated. Thus, from the data in this study, it seems safe to conclude that glycogenin does not play a limiting role in determining maximal glycogen levels in rat skeletal muscle.
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ACKNOWLEDGEMENTS |
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The authors thank Betina Bolmgreen and Irene Bech Nielsen for expert technical assistance.
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FOOTNOTES |
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The present study was supported partly by Grant 504-14 from the Danish National Research Foundation.
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: B. F. Hansen, Diabetes Biology, Novo Nordisk A/S, Bldg. 6B1.56, DK-2880 Bagsvaerd, Denmark (E-mail:bfh{at}novo.dk).
Received 8 March 1999; accepted in final form 13 October 1999.
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