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ABSTRACT |
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1. The influence of muscle glycogen content on glycogen synthase (GS) localization and GS activity was investigated in skeletal muscle from male Wistar rats.
2. Two groups of rats were obtained, preconditioned with a combination of exercise and diet to obtain either high (HG) or low (LG) muscle glycogen content. The cellular distribution of GS was studied using subcellular fractionation and confocal microscopy of immunostained single muscle fibres. Stimulation of GS activity in HG and LG muscle was obtained with insulin or contractions in the perfused rat hindlimb model.
3. We demonstrate that GS translocates from a glycogen-enriched membrane fraction to a cytoskeleton fraction when glycogen levels are decreased. Confocal microscopy supports the biochemical observations that the subcellular localization of GS is influenced by muscle glycogen content. GS was not found in the nucleus.
4. Investigation of the effect of glycogen content on GS activity in basal and insulin- and contraction-stimulated muscle shows that glycogen has a strong inhibitory effect on GS activity. Our data demonstrate that glycogen is a more potent regulator of glycogen synthase activity than insulin. Furthermore we show that the contraction-induced increase in GS activity is merely a result of a decrease in muscle glycogen content.
5. In conclusion, the present study shows that GS localization is influenced by muscle glycogen content and that not only basal but also insulin- and contraction-stimulated GS activity is strongly regulated by glycogen content in skeletal muscle.
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INTRODUCTION |
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Over recent years there has been an increased focus on the significance of spatial compartmentalization of the enzymes involved in glycogen metabolism. The rate-limiting conversion of UDP-glucose to glycogen is catalysed by glycogen synthase (GS), which in skeletal muscle is known to be bound to glycogen particles (Meyer et al. 1970; Bergamini et al. 1977) and myofibrils (Moruzzi et al. 1980; Lane et al. 1989). In 3T3-L1 adipocytes, primary hepatocytes, C2C12 myotubes and COS-1 cells, GS is capable of translocation in response to various stimuli (Fernandez-Novell et al. 1997; Brady et al. 1999; Ferrer et al. 2000). However, tissue-specific regulation of GS translocation in these different cell cultures underlines the importance of investigating GS localization and the functional consequence of this in fully developed skeletal muscle. The glycogen content of skeletal muscle is subject to large changes upon different physiological stimuli. Since GS is bound to glycogen particles it could be hypothesized that GS in situations with low glycogen content (e.g. glycogen-depleting contractile activity) associates with other structures in the muscle cell. Thus, the first aim of the present study was to investigate the influence of muscle glycogen content on GS localization.
A substantial number of studies have investigated the mechanism by which insulin increases glycogen synthase activity (Lawrence & Roach, 1997). Even though the understanding of this mechanism is still incomplete, a substantial number of insulin signalling components involved in glycogen synthesis have been identified. In contrast almost nothing is known about the cellular events leading to increased GS activity following exercise. A negative correlation between glycogen content and GS activity in both basal, insulin- and contraction-stimulated muscles has been demonstrated numerous times (Danforth, 1965; Bogardus et al. 1983; Zachwieja et al. 1991; Munger et al. 1993; Furler et al. 1998), but these correlations do not necessarily prove causality. Thus, our study focuses specifically on the effect of glycogen content on GS activity.
In summary the purpose of the present study was to investigate whether the localization of GS is dependent on glycogen level and whether the activity of GS is influenced by localization of the enzyme. For this purpose we have studied GS in subfractionated rat skeletal muscle and by confocal microscopy of single rat muscle fibres. Furthermore, using the hindlimb perfusion model of rats, we studied the effect of glycogen level on GS activation in basal, insulin- and contraction-stimulated skeletal muscle.
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METHODS |
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Experimental protocol
The experimental protocol of the different experiments included in the study is summarized in Fig. 1. In all experiments the experimental animals were preconditioned between day 1 and day 2 to obtain high (HG) or low (LG) muscle glycogen levels as described below. On day 3 muscle fractionation, confocal microscopy or hindlimb perfusion was initiated and on the following days the different analyses were done.
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The preconditioning regime carried out on day 1 and 2 divided the rats into a high (HG) and a low (LG) muscle glycogen group. Then the different experiments were initiated on day 3 and continued with different analyses on day 4 onwards. See Methods section for a detailed description of the different experimental procedures in the study.
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Experimental animals
All experiments were approved by the Danish Animal Experiments Inspectorate and complied with the Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985). Male Wistar rats (mean weight ± S.E.M. 82 ± 2 g, N = 85) were pre-conditioned in order to obtain two different subgroups with varying muscle glycogen concentrations, as described previously (Hespel & Richter, 1990). Rats were subjected to 2 h of swimming in water maintained at 32-35 °C with weights (5 % of body weight) attached to their tails. In the 24 h preceding the swim, their food intake was restricted to 4 g (~60 % of normal intake). After swimming, all rats had free access to tap water and they were fed ad libitum with either lard (low glycogen, LG), or normal rat chow and a 20 % glucose drinking solution (high glycogen, HG), until 3-6 h before the perfusions were initiated as described below. In the contraction experiment, a control group of rats with normal glycogen concentrations (NG) was not subjected to swimming and received normal chow ad libitum until 3-6 h before perfusions. Rat muscles were excised between 18 and 24 h after the swimming bout.
Hindlimb perfusions
Rats were anaesthetized by an intraperitoneal injection of pentobarbital sodium (5 mg (100 g body weight)-1). Surgery was performed as described by Ruderman et al. (1971) for isolated hindquarter perfusion. Rats were killed by an intracardiac pentobarbital sodium injection. All perfusions lasted 25 min using a constant flow of 6 ml min-1. At the onset of the perfusion, the first 10 ml of the venous outflow were discarded and thereafter the remaining perfusate (90 ml) was recirculated and constantly gassed with a mixture of 95 % oxygen-5 % carbon dioxide. The standard perfusate was cell-free and consisted of Krebs-Ringer bicarbonate buffer solution, 4 % (w/v) bovine serum albumin (BSA; fraction V, Sigma Chemicals, St Louis, MO, USA), 0.15 mM pyruvate, and 4.2 i.u. ml-1 heparin, as previously described (Wojtaszewski et al. 1998). In order to avoid glycogen synthesis during the perfusion period glucose was omitted from the perfusate. For the basal perfusion experiments, hindlimbs were perfused for 25 min with the standard perfusate and muscle samples were taken from both legs immediately after discontinuation of perfusion. The insulin perfusion experiments were identical to the basal perfusions, except that either 100 or 10 000 
U ml-1 insulin (Actrapid, Novo Nordisk, Denmark) was added to the standard perfusate prior to the start of the experiment and was present throughout the 25 min perfusion period. In the contraction perfusion experiments, the standard perfusate was used throughout the experiment. After the first 10 min of perfusion, the left common iliac artery and vein were ligated and muscle biopsies were taken from the left leg (for determination of pre-contraction glycogen levels). After an additional 5 min of equilibration perfusion, the right leg was made to contract isometrically by electrical stimulation of the sciatic nerve for 10 min, whereafter the right leg was biopsied. The electrical stimulation was performed with supramaximal trains (25 V) of 100 ms delivered at 2 s intervals and impulse duration and frequency within the train of 0.1 ms and 100 Hz, respectively. The leg to be stimulated was, from the beginning of the perfusion, immobilized by a pin under the patella tendon and the Achilles' tendon was fixed by a hook pin connected to an isometric muscle tension transducer. Tension developed by the calf musculature was recorded by a pen writer (Clevite Brush Mark 220). The perfusion pressure ranged between 30 and 50 mmHg in basal and insulin perfusions and between 40 and 60 mmHg in contraction perfusions. The muscle samples (10-15 mg) were taken from three different parts of the calf musculature, representing a range of fibre-type distributions, with respective distributions (%) of slow-twitch oxidative, fast-twitch oxidative glycolytic and fast-twitch glycolytic fibres of young rats (60-100 g), taken from Maltin et al. (1989), given in parentheses: the white, most superficial part of the gastrocnemius (0:20:80), the red, deep proximal and medial portion of gastrocnemius (10:55:35) and the soleus (55:40:5). Soleus, red and white gastrocnemius were trimmed of connective tissue, blotted, and freeze clamped with aluminium clamps cooled in liquid nitrogen. The biopsies were stored at -80 °C until analysed.
Low spin preparation
Crude fractionation of muscle for determination of GS activity and GS protein content (see below) was done on soleus and red and white gastrocnemius from LG and HG rats. The samples were homogenized (model OMNI 2000, Omni Int., Warrenton, VA, USA) twice for 15 s in 1 ml ice-cold homogenizing buffer (50 mM Tris, 100 mM NaF and 10 mM EDTA, pH 7.8). This buffer was used for homogenization in all experiments. One half of the homogenate was stored at -20 °C and the other half was centrifuged for 25 min at 2700 g at 4 °C. The supernatant was decanted and frozen and the pellet was resuspended in 0.5 ml homogenizing buffer before storage at -20 °C.
Subcellular fractionation
To further characterize the localization of GS, muscle homogenates from LG and HG rats (see 'Experimental animals') were fractionated by differential centrifugation. The whole gastrocnemius muscle (unseparated red and white gastrocnemius) was homogenized for 30 s in seven volumes of ice-cold homogenizing buffer. The homogenate was centrifuged at 1500 g for 10 min at 4 °C. The post-nuclear supernatant (PNS) was decanted and the pellet was discarded. The PNS was centrifuged at 200 000 g for 45 min at 4 °C. The resulting supernatant containing the majority of the cytosolic proteins was decanted. The pellet was resuspended in 1 % Triton X-100 in homogenizing buffer and incubated at 4 °C with end-over-end rotation for 30 min. The suspension was centrifuged at 17 000 g for 5 min and the resulting supernatant and pellet were termed membrane fraction and cytoskeleton fraction, respectively. The different fractions were subjected to SDS-PAGE. GS protein content and actin content (used as a cytoskeleton marker) were determined by Western blotting (see below). GS activity in the different fractions was measured as described below.
Western blotting
GS and actin protein content were determined by Western blotting. All samples were separated on a 10 % SDS-polyacrylamide gel followed by protein transfer to a PVDF membrane by semi-dry electro-blotting as previously described with minor modifications (Kristiansen et al. 1996). The membrane was blocked in 5 % (w/v) BSA in TBS-T (10 mM Tris, 0.9 % (w/v) NaCl, 0.1 % (w/v) Tween-20, pH 7.45) for 1 h at room temperature before overnight incubation with a rabbit anti-GS serum (donated by Oluf Pedersen, Steno Hospital, Gentofte, Denmark) diluted 1: 20 000 in 2 % BSA in TBS-T. After two 10 min washes in TBS-T, the membrane was incubated for 1 h with an anti-mouse AP-conjugated antibody (Zymed Laboratories Inc., San Francisco, CA, USA) for 1 h. The membrane was washed twice for 20 min each time in TBS-T before a chemifluorescence substrate (Vistra ECF, Amersham Int., Uppsala, Sweden) was applied to the membrane. The resulting signal was detected by a STORM scanner (Amersham Int.) and quantified with ImageQuant software (Amersham Int.).
GS localization in teased single fibres by confocal microscopy
Muscle fixation and staining of single fibres was carried out essentially as described by Ploug et al. (1998). In short, rat hindlimb muscles were fixed by perfusion with 2 % depolymerized paraformaldehyde containing 0.15 % picric acid. The extensor digitorum longus (EDL) muscle was removed and fixed for an additional 4 h at 4 °C and then transferred to PBS. Bundles of one to three individual fibre fragments were teased from fixed muscle with fine forceps, permeabilised with 0.03 % saponin and labelled overnight with rabbit anti-GS serum diluted 1: 500. The primary antibody was visualized with a fluorescein isothiocyanate (FITC)-labelled secondary antibody. Fibres were mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA) and confocal images collected on a Zeiss LSM 410 microscope at the NINDS Light Imaging Facility (Bethesda, MD, USA). Settings were adjusted so that images of fibres stained without primary antibody appeared essentially black. Images were adjusted for contrast with Adobe Photoshop software (Adobe Systems, Inc., San Jose, CA, USA).
Glycogen and GS activity measurements
Muscle glycogen content was measured as glucose residues by a hexokinase method after acid hydrolysis (Passonneau & Lowry, 1993). GS activity was measured in triplicate on 20 l of each sample by a modification of the method of Thomas et al. (1968) as described previously (Richter et al. 1989). Synthase activity at a maximally stimulating glucose 6-phosphate concentration of 8 mM is defined as total activity because the enzyme is fully activated, regardless of its phosphorylation status. The activity at a glucose 6-phosphate concentration of zero divided by the total activity is defined as the independent form (% I-form), whereas the activity measured at 0.17 mM glucose 6-phosphate divided by the total activity is defined as fractional velocity.
Statistics
In order to test differences between pre- and post-contraction samples, Student's paired t test was used. When analysing effects of glycogen and insulin and their interaction, two-way ANOVA was used. Data are presented as means ± S.E.M. and 0.05 was chosen as the level of significance.
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RESULTS |
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Effect of preconditioning regime on muscle glycogen content
The content of glycogen was measured in muscles used in the various experiments. In the subcellular fractionation experiments the whole gastrocnemius muscle was used (unseparated red and white gastrocnemius). The overnight feeding of rats with a carbohydrate-rich diet (HG group) compared to a carbohydrate-free diet (LG group) following a glycogen-depleting swimming exercise bout, resulted in a ~4-fold higher glycogen content in the gastrocnemius muscle (pooled data from the three experiments) (data not shown). In the microscopy experiments the epitrochlearis muscle (with a fibre-type distribution resembling EDL) was used for determination of glycogen content because the method utilized for fixation of the rat hindlimb made glycogen determinations unattainable in EDL muscle. HG epitrochlearis had a ~3-fold higher glycogen content than LG epitrochlearis (data not shown). In the insulin experiment, the glycogen content of the HG group was from 4.6- to 6.6-fold higher (HG vs. LG, P < 0.05) in all types of muscles investigated, with the highest glycogen concentration in red gastrocnemius muscles from HG rats (Table 1). For the contraction experiment we obtained a group of non-exercised, chow-fed rats with normal glycogen (NG), in addition to the high (HG) and low glycogen (LG) groups. Before contractions, the NG group had muscle glycogen levels 1.4-fold higher than the LG group for both red and white gastrocnemius, and 2.3- and 3.6-fold lower than the HG group for white and red gastrocnemius, respectively (Table 2). After contractions the NG group had muscle glycogen levels 2.2- and 2.8-fold higher than the LG group, and 2.9- to 3.1-fold lower than the HG group (Table 2). Contractions decreased the glycogen level by, respectively, 24 and 38 % in the HG white and red gastrocnemius and 59 and 63 % in the LG white and red gastrocnemius. Data from soleus are not presented since, as previously demonstrated (Derave et al. 1999), the submaximal stimulation protocol only resulted in a minimal glycogen breakdown in this muscle.
Total GS activity and GS protein content in homogenates
A maximal saturating concentration (8 mM) of glucose 6-phosphate present in the GS activity assay fully activates the enzyme, regardless of its phosphorylation status and is therefore thought to represent the total GS protein content present in the homogenate. As shown in Table 1 (data pooled from basal and insulin-stimulated muscles), the total GS activity measured in crude homogenates in HG muscles was significantly higher than in LG muscles. Western blot analysis for GS protein content (Table 1), however, failed to show a significant difference in GS protein levels between HG and LG muscles. This shows that total GS activity does not fully correspond to total GS protein content as determined by Western blotting. However, independent of glycogen content, both total GS activity and GS protein content were significantly higher in the red than in the white portion of the gastrocnemius, with intermediate values in soleus (Table 1).
GS activity and GS protein in low spin fractions
GS activity in skeletal muscle samples is conventionally measured in supernatants after a low-speed centrifugation of homogenates. We have now measured the distribution of GS activity in the pellet and supernatant of a homogenate subjected to a 2700 g centrifugation (20 min) which provides a crude subcellular fractionation. The pellet contains mainly nuclei, cytoskeleton and membrane proteins, whereas the supernatant contains cytosolic and membrane-associated proteins as judged by Western blotting with antibodies against marker proteins for membranes (GLUT4) and the cytoskeleton (actin) (data not shown). In white and red gastrocnemius and soleus, respectively, 33, 30 and 24 % of the total GS activity was recovered in the pellets from the HG group, whereas these activities were 54, 62 and 27 % in pellets from the LG group (P < 0.05 vs. HG, in white and red gastrocnemius) (Fig. 2). In red gastrocnemius it was verified that the distribution of total GS activity corresponded to a similar distribution of GS protein (Fig. 3). Furthermore, Western blot analysis showed that the GS protein content of the supernatants decreased as the glycogen level decreased (Fig. 4), whereas the total GS protein content measured in homogenates was independent of the muscle glycogen content (Table 1). The GS activities measured in muscle homogenates in the presence of 0 and 0.17 mM glucose 6-phosphate, expressed relative to the total GS activity, are called, respectively, the independent form (% I-form) and the fractional velocity of GS activity (see Methods). The fractional velocity of GS recovered from pellet and supernatant is shown in Fig. 5. In all muscle types, the GS found in the supernatants of supercompensated (HG) muscles showed slightly but significantly lower fractional velocities than the GS found in the pellets. In glycogen-depleted muscles (LG), this was only true for the soleus, and not for the gastrocnemii. The % I-form of the GS in the pellet and supernatant were not significantly different from each other (data not shown).
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Data pooled from white and red gastrocnemius muscle.
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Intracellular localization of GS protein
To study further the intracellular localization of GS, a post-nuclear supernatant (PNS) was prepared from HG and LG gastrocnemius muscle (unseparated red and white gastrocnemius). Soleus muscle was not used since GS localization in this muscle was unaffected by glycogen level, as judged from the low spin preparation (Fig. 2). The pellet from this 1500 g spin contains mainly nuclei and large cellular debris. Because it has been suggested that GS may reside in the nuclei of various cell lines in culture in the absence of glucose (Ferrer et al. 2000), we purified nuclei from glycogen-depleted gastrocnemius muscle, but found no GS associated with the nuclei (data not shown). The PNS from HG and LG gastrocnemius muscle contained equal amounts of GS (Fig. 6). Further fractionation of the PNS showed that more GS is found in the cytoskeleton fraction of LG muscle than HG muscle, whereas the membrane fraction of HG muscle contained more GS than the membrane fraction of LG muscle. No cytosolic GS protein was detectable in HG or LG muscle. Evaluation of the amount of actin (used as a cytoskeleton marker) in the different fractions demonstrates that actin was almost exclusively present in the cytoskeleton fraction and that the actin content was similar in the different LG and HG fractions (Fig. 6). Evaluation of total GS activity in the different fractions confirmed the GS protein data obtained by Western blotting. Fractional velocity (Fig. 6) and % I-form (not shown) were ~2- and ~4-fold higher, respectively, in the LG fractions than in the HG fractions (PNS, membrane and cytoskeleton); thus the degree of GS activation was independent of GS localization. In both HG and LG muscle, glycogen was almost exclusively present in the membrane fraction (data not shown).
To obtain direct evidence for glycogen-dependent subcellular localization of GS, teased single EDL fibres were stained with an antibody against GS. As seen from Fig. 7, the immunofluorescence staining shows two components: a cross-striated pattern, most probably associated with the I-bands, and a broader staining in the perinuclear areas. Both are present in all fibres, irrespective of glycogen level. The fine structure of the staining, however, shows striking differences between LG and HG fibres. In low glycogen muscle, the staining has a fine granular structure which gives the cross-striations a smooth, continuous appearance. In high glycogen fibres, in contrast, the staining appears coarse, suggesting larger aggregates of the protein, as well as areas without protein. The morphological data thus support the biochemical observations that the subcellular localization of GS is influenced by muscle glycogen content.
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A representative Western blot of subfractionated gastrocnemius muscle (mixed red and white) from rats with low (LG) and high (HG) muscle glycogen content (upper panel). The different fractions (see Methods section) were run on 10 % SDS-PAGE and incubated with a GS antibody and an actin antibody as a cytoskeleton marker. PNS, post-nuclear supernatant. Total GS activity (middle panel) and GS fractional velocity (%) (lower panel) were measured in the different fractions with low (
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Different staining patterns are observed for glycogen synthase in fibres with low (left) and high (right) glycogen. Single fibres were prepared from extensor digitorum longus muscles with either low or high glycogen levels and stained with an antibody against glycogen synthase. Confocal images were collected at the surface of the fibres. The arrows point to the areas which are shown enlarged in the insets. Note that the cross-striated glycogen synthase staining in fibres with low glycogen appears distinct and continuous in contrast to a dotted appearance in fibres with high glycogen. Bar: 10
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Effect of glycogen level on insulin-stimulated GS activity
There was a significant effect of the muscle glycogen content on GS fractional velocity in all muscle types (Fig. 8). Unstimulated (0 U insulin (ml perfusate)-1) GS fractional velocity (measured on crude homogenates) ranged from 4 to 13 % in HG muscles and from 44 to 56 % in LG muscles (Fig. 8A-C). Insulin (100 and 10 000 U ml-1) caused stepwise significant increases in GS fractional velocity in the gastrocnemius muscles. A similar tendency was observed in soleus but was, however, statistically insignificant. The maximally insulin-stimulated GS fractional velocity in HG was still significantly lower than the unstimulated value in LG in all muscles (Fig. 8). There was a significant effect of glycogen on GS % I-form in all muscles and a significant effect of insulin in white gastrocnemius and soleus. There was a significant interaction between the effects of glycogen and insulin on GS % I-form in both white and red gastrocnemius. This is illustrated in Fig. 8D and E where insulin has no effect in HG muscle, whereas in LG muscle insulin has a large effect.
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Glycogen synthase (GS) fractional velocity is shown in the left panel (A-C) and GS % I-form in the right panel (D-F). Measurements were done on crude homogenates on white gastrocnemius (A, D), red gastrocnemius (B, E) and soleus (C, F) with high (HG) or low (LG) glycogen contents. Hindlimbs were perfused in the presence of 0 (
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From Fig. 9, it can be seen that GS fractional velocity is dependent upon the glycogen content in an inverse non-linear manner at all three concentrations of insulin. The best-fit lines for the pooled group of muscles at the three insulin concentrations are shown and it can be seen that insulin shifts the relationship between glycogen content and GS fractional velocity to a slightly different curve, but does not abolish the relationship, indicating that glycogen is a more potent regulator of GS activity than insulin.
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U ml-1 insulin
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Effect of glycogen level on contraction-stimulated GS activity
Figure 10 shows the effect of 10 min of electrically stimulated contractions on GS activity (fractional velocity and % I-form). The measurements were done on homogenates of perfused gastrocnemius muscles with high (HG), normal (NG) or low (LG) glycogen content. In white gastrocnemius muscles, the contraction-induced changes in GS fractional velocity (%) and % I-form, respectively, were +2 % and -1 % in HG (not significant, n.s.), +37 % and +8 % in NG (P < 0.01) and +22 % (P < 0.05) and +5 % (n.s.) in LG. In red gastrocnemius muscles, these changes are 11 % (P < 0.05) and 0 % (n.s.) in HG, 25 % and 8 % in NG (P < 0.01) and 22 % and 12 % in LG (P < 0.01), respectively. From Fig. 11, it can be seen that in unstimulated muscles, GS fractional velocity is dependent upon the glycogen content in an inverse non-linear manner. The best-fit line for pre-stimulation samples is shown, and followed the equation y = 3900x-1.417. The average deviation from this line was 2.5 % for the pre-stimulation samples and -4.6 % for the post-stimulation samples (post-stimulation values with glycogen < 13 mol (g wet wt)-1 had predicted fractional velocities of > 100 %; therefore the predicted value was set at 100 % for these samples). Thus, the contraction-stimulated samples fitted the same equation line as the resting muscle, indicating that the contraction-induced GS fractional velocity is merely a result of a decrease in muscle glycogen content. The same interpretation holds for % I-form (data not shown). Force development by the calf musculature (maximal and mean force) was unaffected by the variation in glycogen content in the three experimental groups of rats (Table 3).
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the present study we have investigated the effect of glycogen content on the activity and intracellular localization of GS in skeletal muscle. We demonstrate for the first time that the GS protein translocates from a glycogen-enriched membrane fraction to the cytoskeleton when glycogen levels are decreased. Furthermore, we demonstrate that glycogen is a far more potent regulator of GS than insulin. Finally, the stimulatory effect of muscle contractions on GS activity is solely a function of the ability of contractions to decrease muscle glycogen concentration.
It has previously been demonstrated that in skeletal muscle GS is associated with both glycogen particles (Meyer et al. 1970; Bergamini et al. 1977) and myofibrils (Moruzzi et al. 1980; Lane et al. 1989), but we show, for the first time, that GS translocates from the glycogen-enriched fraction to the cytoskeleton when glycogen content in skeletal muscle decreases (Fig. 6). Interestingly, it has been demonstrated (Baque et al. 1997) that the GS subunit glycogenin is partly associated with the cytoskeleton protein actin, which constitutes a major part of the sarcomeric I-band. In the early stages of glycogen synthesis, glycogenin and GS are associated, but later they dissociate, as GS is thought to move to the outer branches of the glycogen particle (Smythe & Cohen, 1991). If GS activity is high only when glycogenin and GS are bound to each other as suggested by in vitro studies (Pitcher et al. 1987, 1988), this could explain why GS activity decreases as the glycogen particle grows. Differences in GS activity or susceptibility to activation when bound or unbound to glycogen have been hypothesized (Lane et al. 1989) and could suggest a link between the differences in localization and activity of GS in muscle with high and low glycogen presented here. This is supported by our finding that in most situations the 2700 g supernatants showed significantly lower fractional velocities compared to the GS found in the pellets (Fig. 5). In our fractionation assay, however, the activity ratio of GS in the different cell fractions (Fig. 6) is not affected by localization in the sense that when glycogen is low the activity ratio of GS is high independently of subcellular localization. Conversely, when glycogen is high GS activity is low independently of localization. Nevertheless, even though the activity ratio of GS in a muscle with a given glycogen content is quite similar in the various subcellular fractions, the change in subcellular localization of GS with different glycogen concentrations may have important functional consequences. For instance, the preferential cytoskeletal localization of GS when glycogen stores are depleted probably indicates that glycogen synthesis close to the contractile apparatus has a high priority. It is presumably advantageous to have the glycogen stores located close to the site of energy utilization especially because mitochondria also accumulate in this region (Eisenberg et al. 1974; Eisenberg & Kuda, 1975). When glycogen stores near the contractile apparatus are filled up then further glycogen synthesis can occur in other regions of the cell and GS association with the myofibrils is relaxed (Fig. 7). Furthermore, it might be speculated that the subcellular localization of GS is important for the ability of insulin to activate GS. If so this would offer an explanation for the variation in insulin's ability to activate GS depending on glycogen concentration (Fig. 8). Finally, it could be argued that the hypothesized increased susceptibility to activation of GS bound to the cytoskeleton is dependent on intact in vivo conditions and as such not fully reflected in the in vitro GS activity assay.
Even though the functional consequences of the GS translocation in response to changes in glycogen content remain to be clarified, the activity of GS is clearly dependent on glycogen level. This is in accordance with a number of previous studies (Danforth, 1965; Bogardus et al. 1983; Richter et al. 1988; Zachwieja et al. 1991; Munger et al. 1993; Furler et al. 1998). However, it is of note that when glycogen levels are low, GS activity in unstimulated muscle is higher than even maximally insulin-stimulated GS activity in HG muscle, indicating that glycogen is a more potent regulator of GS activity than insulin. This is shown in Fig. 8, where it is illustrated that insulin has a markedly smaller effect on GS % I-form in muscle with high glycogen than in muscle with low glycogen. Interestingly, insulin-stimulated glycogen synthesis in human muscle is also negatively influenced by glycogen content (Laurent et al. 2000), supporting the notion that glycogen inhibits its own synthesis. Prior exercise is known to increase GS activity in response to insulin stimulation (Richter et al. 1984; Wojtaszewski et al. 2000). The observations in the present study suggest that the period of glycogen depletion following an exercise bout is at least in part responsible for this increased effect of insulin. This could be due to regulation of the insulin signalling proteins upstream of GS. Whereas the initial signalling intermediates (insulin receptor tyrosine kinase (IRTK) and phosphoinositide-3 kinase (PI3K)) are not influenced by muscle glycogen content (Goodyear et al. 1995; Wojtaszewski et al. 1997; Derave et al. 2000), the effect of insulin on the more downstream protein kinase B (PKB) is enhanced by lowering glycogen levels (Derave et al. 2000). Currently, we are investigating whether the glycogen level modulates the insulin-induced deactivation of glycogen synthase kinase 3 (GSK3) activity, which is thought to act downstream of PKB (Cross et al. 1995, 1997; Lawrence & Roach, 1997; Ueki et al. 1998) and upstream of GS (Cross et al. 1997; Lawrence & Roach, 1997; Ueki et al. 1998). Interestingly, early in vitro studies have shown that physiological concentrations of glycogen were found to inhibit the conversion of GS in the D-form into the active I-form, suggesting inhibition of GS phosphatase activity (Villar-Palasi & Larner, 1966; Villar-Palasi, 1969). The responsible phosphatase could be protein phospatase 1 (PP1) since PP1 is capable of dephosphorylating GS (Cohen, 1993; Ragolia & Begum, 1998). One possible way that glycogen might affect GS activity could be through interaction with the glycogen targeting subunits of PP1 which bind the catalytic subunit of PP1 to glycogen and also bind GS, glycogen phosphorylase and phosphorylase kinase (for review see Newgard et al. 2000). The binding of GS to the glycogen targeting subunits of PP1 is apparently important for activity of GS (Newgard et al. 2000). In skeletal muscle the major glycogen targeting subunit expressed is termed GM or RGL and this subunit of PP1 thus serves as a molecular scaffold, binding phosphorylase and phosphorylase kinase in addition to GS and the catalytic subunit of PP1 at the glycogen granule (Newgard et al. 2000). The extent to which glycogen concentration affects binding of GS to GM/RGL is, however, not known. Future studies will have to clarify whether the effect of glycogen content on GS activity is exerted via differences in binding of GS to the glycogen targeting subunits of PP1.
Muscle contractions increase GS activity (Kochan et al. 1979; Richter et al. 1984; Bak & Pedersen, 1990; Brau et al. 1997; Huang, 1998) but, in contrast to the insulin signalling pathway leading to activation of GS, almost nothing is known about the mechanism by which exercise affects GS activity. The present and previous studies demonstrate that exercise-induced GS activity correlates negatively with glycogen content (Danforth, 1965; Zachwieja et al. 1991) and our findings suggest that the increase in GS activity, induced by muscle contractions, is merely a result of the decreasing glycogen content (Fig. 11). In other words, whichever signalling pathway is induced by muscle contractions it does not have an independent effect on GS activity. Moreover there seems to exist a critical glycogen concentration around 50 mol (g wet wt)-1 above which GS in rat muscle does not get activated by contractions (Fig. 11). It is noteworthy that the results obtained in the non-exercised and non-dietary-manipulated control (NG) group were well within the data obtained in the two groups with extreme muscle glycogen levels (Fig. 10). This is an important observation, because it signifies that the differences in contraction-induced GS activity between the groups are not caused by some non-specific effect of the combination of exercise and diet but rather are genuinely related to differences in glycogen content. It seems paradoxical that there exists a mechanism which upregulates glycogen synthesis in glycogen-depleted, working muscle as seen in the present and other studies (Constable et al. 1984; Blom et al. 1986; Vøllestad et al. 1989; Price et al. 1991). From a teleological point of view one would expect that during contractions all available glucose and ATP is directed towards fuelling the contractions instead of being utilized as substrates for glycogen storage. However, it may also be part of a functional mechanism of 'glycogen sparing' in the exercising muscle, protecting it against complete glycogen depletion during intense exercise or early glycogen depletion during prolonged exercise. In addition, one clear advantage of an increased glycogen synthase activity during exercise is that the muscle cell is primed for glycogen synthesis immediately at the cessation of exercise.
In conclusion, the present study shows that glycogen exerts a strong influence on the unstimulated GS activity as well as on the ability of both insulin and muscle contractions to increase GS activity. Our data suggest that the exercise-induced increase in GS activity is entirely a result of decreasing glycogen level. The insulin effect on GS activity is improved in muscles having a low level of glycogen, pointing towards an upregulation of the activity of insulin signalling intermediates. The localization of GS could be important in explaining the tight association between glycogen level and GS activity, since we find that GS translocates from the glycogen-enriched membrane fraction to the cytoskeleton as glycogen content is lowered in the muscle cell. Subcellular translocation of GS may thus be important for the regulation of GS activity.
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Acknowledgements
This study was supported by grant no. 504-14 from the Danish National Research Foundation. The authors are grateful to Betina Bolmgren and Jozef Langfort for superior technical contributions.
J. N. Nielsen and W. Derave contributed equally to this work.
Corresponding author
E. A. Richter: Copenhagen Muscle Research Centre, Department of Human Physiology, University of Copenhagen, 13 Universitetsparken, DK-2100 Copenhagen, Denmark.
Email: erichter{at}aki.ku.dk
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, pellet; 
, supernatant. Sum of activities measured in pellet and supernatant is set as 100 %. Number of observations in each condition is indicated in parentheses above each bar. GW, white gastrocnemius; GR, red gastrocnemius; Sol, soleus. * Significantly different from HG.
and 
, rats with high muscle glycogen; 
and 
, rats with low muscle glycogen; 
) and 10 000 
, 100