Regulation of glycogen accumulation in L6 myotubes cultured under optimized differentiation conditions

Peter Elsner, Bjørn Quistorff, Thomas S. Hermann, John Dich, and Niels Grunnet

Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The differentiation of the L6 myogenic cell line was enhanced by the addition of dexamethasone, retinoic acid, insulin-like growth factor I (IGF-I), and creatine. Spontaneous contractions appeared from day 10 or 11 and persisted to day 14 or 15. Glucose transport was increased by insulin (100 nM) and IGF-I (5 nM) by ~60%. The highest level of glycogen was measured in myotubes differentiated under the influence of a combination of 5 nM dexamethasone, 100 nM retinoic acid, 5 nM IGF-I, and 10 mM creatine with glucose as substrate. The glycogen accumulation rate was constant from 0 to 2 h of incubation and decreased gradually to zero at 4 h. From 0 to 0.5 h of the glycogen accumulation, the glycogen synthase a (GSa) activity was 30-35% of the total activity, with a subsequent gradual decline to 2.5% after 6 h. The glycogen phosphorylase a (GPha) activity was constant at ~80% from 0 to 0.5 h, increasing to ~100% after 6 h. The activity ratio of GSa to GPha decreased about sixfold without significant change in the rate of glycogen accumulation. This indicates that factors other than phosphorylation/dephosphorylation play a decisive role in the regulation of glycogen metabolism in L6 myotubes. Intracellular glucose (glucosei) and glucose 6-phosphate (G-6-P) may be such factors. The observed values of these parameters may in fact explain an activation of GSa (G-6-P) and an inhibition of GPha (glucosei).

muscle; cell culture; creatine kinase; glycogen metabolism; cell differentiation

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE PRESENT STUDY is concerned with the differentiation and glycogen metabolism of the myogenic cell line L6 developed by Yaffe in 1968 (56). The L6 myotubes exhibit several myogenic characteristics, i.e., contractility (50), capacity to form myoneural junctions (51), resting membrane potentials of about -70 mV (23), acetylcholine receptors (41), synthesis of actinomyosin (30), insulin-sensitive glucose transport by the GLUT-4 transporter (33), active lactate transport (6), and expression of creatine kinase, albeit at low levels (44). However, the L6 myotubes also lack certain skeletal muscle characteristics, such as a high ratio of phosphocreatine to ATP (PCr/ATP) (32), high glycogen levels (36), and a low intracellular glucose concentration (17). Several differentiation factors have been reported to enhance myogenic differentiation of L6 cells (1, 7, 11, 15, 49, 55), but typically the effect of individual factors has been studied. Only a few studies have dealt with the effect of combinations of differentiation factors (55), and thus the possibility of more complete differentiation of muscle-specific characteristics of the cell line has not been explored. For example, the ability of L6 myotubes to accumulate glycogen is poorly investigated. Only one study on the effect of medium glucose concentration on glycogen accumulation (54) and two studies concerned with the effect of insulin on glycogen accumulation (26, 42) have been reported. However, none of these involved addition of differentiation factors.

The aims of the present investigation, therefore, were 1) to enhance the differentiation level of L6 cells by combinations of differentiation factors, including factors that have been reported to increase the expression of GLUT-4, 2) to identify conditions that stimulate glycogen accumulation, and 3) to study the regulation of glycogen metabolism under these conditions of optimized differentiation.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials

L6 cells (L6.G8.C5) were obtained from the European Collection of Animal Cell Cultures and were routinely used after three passages. Fetal calf serum (FCS) was from Biological Industries. Penicillin was from Løvens Kemiske Fabrik. Insulin-like growth factor I (IGF-I) was a gift from Pharmacia. Dexamethasone (Dex) was from Merck, Sharp & Dohme. Insulin (porcine) was from Novo Nordisk. Culture dishes were from Nunc. 3-O-methyl-D-[1-3H]glucose, [U-14C]glucose 1-phosphate, and UDP-[U-14C]glucose were from Amersham Radiochemical Center. SeaPlaque agarose was from FMC BioProducts. UDP-glucose, glucose 1-phosphate, glucose 6-phosphate (G-6-P), and glycogen were from Boehringer Mannheim. Trypsin was from GIBCO-BRL. DMEM, gentamicin, creatine (Cr), retinoic acid (Ret.a), triiodothyronine (T3), cytosine-1-beta -D-arabinofuranoside (AraC), and all other chemicals were from Sigma Chemical.

Cell Culture

L6 myoblasts (56) were plated in 35-mm-diameter (glucose transport experiments) or 60-mm-diameter (all other experiments) uncoated petri dishes at a density of ~4,500 cells/cm2 and incubated at 37°C in 5% CO2-95% air in a medium composed of 10% (vol/vol) FCS and DMEM with 5 mM glucose until confluence occurred (7 days). Penicillin and gentamicin were added to DMEM to a concentration of 100 IU/ml and 50 µg/ml, respectively. The medium was changed every 48 h. From day 7 to day 10, the medium was changed to DMEM with 2% (vol/vol) FCS, different combinations of differentiation effectors, and 10 µM AraC to prevent further proliferation of nonfused mononucleated myoblasts (53). From day 10 to the day of the experiment, the medium remained the same, except that AraC was excluded.

Analytic Procedures

Preparation of homogenates for measurement of enzyme activities and protein and DNA content. The medium was removed by suction from the dishes, and the cells were washed three times at 1-2°C with Hanks' balanced salt solution (HBSS). Glycyl-glycine buffer (1,200 µl) (14) was added to each dish, and the cells were scraped loose with a rubber policeman and homogenized with the dish on ice by ultrasonication (Branson Sonifier B-12) at 50 W for 5 s. The homogenates were kept frozen at -80°C until analyzed.

Preparation of extracts for measurement of ATP and PCr. One dish at a time was placed on ice, the medium was quickly removed by suction, and 1,000 µl of ice-cold 0.7 M perchloric acid (PCA) were added immediately. The cells were scraped loose with a rubber policeman, and aliquots of the extract were kept frozen at -80°C until analyzed.

Preparation of extracts for measurement of G-6-P and intracellular glucose. One dish at a time was placed on ice, the medium was quickly removed by suction from the dishes, the cells were washed three times at 1-2°C with 2 ml HBSS containing 0.4 mM phloretin (from freshly made stock solution of 100 mM phloretin in 100% DMSO), and immediately after this 500 µl of ice-cold 1.2 M PCA were added. The cells were scraped loose with a rubber policeman, and aliquots of the extract were kept frozen at -80°C until analyzed. It was demonstrated that this washing procedure removed >99.8% of the medium glucose.

Preparation of extracts for measurement of glycogen. The medium was removed from the dishes by suction, and 1,000 µl of 0.4 N KOH were added to each dish. After 5 min at room temperature, aliquots of the homogenates were stored frozen at -20°C until analyzed.

Measurements

Enzymes. Creatine kinase (CK) activity was measured spectrophotometrically as described by Passonneau and Lowry (40). Glycogen synthase (GS) activity was measured radiochemically, as described by Golden, Wals, and Katz (13), at 37°C with an incubation time of 60 min. Glycogen phosphorylase (GPh) was measured radiochemically at 37°C in the direction of glycogen synthesis, as described by Gilboe et al. (12), except that Dowex columns replaced filter papers. The specifications of these columns and the elution procedure were as in the GS assay (13). Incubation time was 60 min.

Glucose transport. 3-O-Methyl-D-[1-3H]glucose uptake was measured at 25°C in 35-mm culture dishes. Before the uptake measurement, the dishes were prewashed three times at 25°C with 2 ml of buffer composed of 122 mM NaCl, 4.9 mM KCl, 2.5 mM MgSO4, 36 mM Tris · HCl, pH 7.4, and 1 mM CaCl2 (25). 3-O-Methyl-D-[1-3H]glucose uptake was measured over a 30-s interval in the same buffer + 10 µM 3-O-methyl-D-[1-3H]glucose (3 µCi/ml), 100 nM Ret.a, 5 nM Dex, and 10 mM Cr, with or without 5 nM IGF-I and with or without 100 nM insulin. Nonspecific uptake was determined in the presence of 10 µM cytochalasin B, and the value was subtracted from the total uptake. After the uptake period, the dishes were washed three times at 1-2°C with 2 ml of the prewashing buffer described above + 1 mM HgCl2, the cells were disrupted in 1,000 µl of 0.4 N KOH, and cellular radioactivity was counted by liquid scintillation.

Protein. Protein content was measured by the method of Lowry et al. (28) with bovine serum albumin as standard.

DNA. DNA content was measured by the method of Kissane and Robins (24).

PCr and ATP. PCr and ATP were measured spectrophotometrically as described by Passonneau and Lowry (38).

G-6-P and glucosei. G-6-P and glucosei were measured fluorometrically as described by Passonneau and Lowry (39).

Glycogen. Glycogen was determined as glucose after treatment with amyloglucosidase, as described by Katz et al. (21).

Light microscopy. The cells were examined each day by a Leitz Diavert phase-contrast microscope to evaluate the degree of differentiation.

Test of contractility. Contractility was evoked by a Digitimer D330 Multistim electrostimulator connected to the electrodes. Electrodes were constructed as described by Rösler et al. (43), with an electrode distance of 45 mm. The 4% agar in HBSS used by Rösler was replaced by 2% SeaPlaque agarose in DMEM. The cultures were stimulated for 10 s with 4 V/cm electrode distance in pulse trains of 500 ms at 100 Hz with a 2-ms pulse width. The interval between pulse trains was 500 ms.

Statistics

Data were analyzed by ANOVA and Tukey's procedure. Significance was accepted at P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Differentiation

The L6 myoblasts reached confluence at day 6. At day 7 the cells aligned, and scattered small myotubes were observed (Fig. 1A). When medium was changed to fusion medium with FCS content of 2%, the fusion process accelerated until day 10 or 11, when most of the myoblasts were fused into multinucleated myotubes (Fig. 1, B and C). The nuclei remained in clusters and did not display any lateral displacement in the myotube. The subclone and passage used for the present experiments showed spontaneous contractions in the interval between days 10 and 15. Visible (light microscopy) cross-striation appeared one day after the first spontaneous contractions could be observed (Fig. 1C). From day 14 to day 15, cells began to degenerate. This in particular was the case for myotubes with added Dex.


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Fig. 1.   L6 myotubes during differentiation. A: 7-day-old myoblasts fusing into small myotubes just before fusing process was accelerated. B: myotubes at day 11 cultured without differentiation factors + 10 mM creatine (Table 1). C: cross-striated myotubes at day 11 differentiated with 100 nM retinoic acid, 5 nM insulin-like growth factor I (IGF-I), and 10 mM creatine (Table 1). Scale bars, 100 µm.

Effect of differentiation factors. The specific activity of CK was used as a marker of myogenic differentiation. Effectors like T3, Dex, IGF-I, insulin, Ret.a, and Cr, which have previously been ascribed potential as myogenic differentiators (1, 7, 11, 15, 16, 31, 49, 55), were tested in screening experiments either individually or in combinations from the day of onset of fusion of the myoblasts (day 7) to obtain the most optimal culture conditions for later experiments.

The largest effect on differentiation of a single factor was observed with 5 nM IGF-I or 100 nM insulin (results not shown). IGF-I showed a positive effect (~25%) on CK from day 7 to day 14. The effect of insulin was similar to the effect of IGF-I, but its presence during a longer period (>4 days) caused more cell debris than IGF-I (results not shown). When 100 nM retinoic acid was added in combination with 5 nM IGF-I (Table 1), CK was at the same level as with IGF-I alone. However, the myotubes appeared morphologically more uniform with respect to thickness and length. Addition of 5 nM T3 showed no effect on CK activity alone or in combination with other effectors (results not shown).

                              
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Table 1.   Specific activity of CK in L6 cells during differentiation

In 10-day-old cultures, 5 nM Dex inhibited differentiation (Table 1), and when added to the combination of Ret.a and IGF-I, it abolished their effect on CK activity, which decreased to control level (Table 1). In 14-day-old cultures, Dex had no effect on differentiation (Table 1). However, the myotubes appeared morphologically more degenerated as judged by light microscopy.

Effect of CR. Addition of 10 mM Cr from day 7 caused an increase in the differentiation level. The largest effect was observed with the low CK levels, i.e., with 5 nM Dex, when Cr increased CK by 64 and 38% at days 10 and 14, respectively (Table 1). The lowest effect was seen when the CK activity was already high, i.e., 5 nM IGF-I + 100 nM Ret.a, when Cr gave rise to an increase of 22 and 18% at days 10 and 14, respectively (not significant at day 14; Table 1). Myotubes cultured under this condition showed the highest degree of differentiation [7.8 and 9.8 U of CK/mg protein at days 10 and 14, respectively (Table 1)]. PCr/ATP in L6 myotubes is reported to be rather low compared with that in the intact muscle (~4) (32). This was confirmed in the present study (Table 2). Addition of 10 mM Cr to the medium, however, resulted in a >10-fold increase of PCr/ATP from 0.24-0.28 to 2.8-4.1 (Table 2). Cr concentrations of 0.1 or 1.0 mM were without significant effect on PCr/ATP.

                              
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Table 2.   Effect of medium Cr addition on PCr/ATP ratio in L6 cells

Contractility. The subclone and passage used for the present experiments showed spontaneous contractions from days 10-11 to days 14-15. However, contractions were not coordinated in time or across the dish. When exposed to electrical stimulation, one-third to one-half of the myotubes were able to contract in concert. The earlier the appearance of spontaneous contractions was observed, the higher was the differentiation level, i.e., IGF-I + Ret.a + Cr. From day 14 to day 15, the tubes became too degenerated to contract either spontaneously or by electric stimulation.

As a consequence of the above results, further experiments were carried out with 5 nM IGF-I, 100 nM Ret.a, and 10 mM Cr from day 7.

Glycogen Accumulation and Glucose Transport

L6 myotubes cultured at control conditions are able to accumulate only low levels of glycogen (18, 37, 54). In screening experiments, combinations of different medium glucose concentrations (5, 10, and 25 mM) and insulin concentrations (0, 10, and 100 nM) were tested for effects on glycogen accumulation over 24 h on 14-day-old myotubes in the absence of IGF-I, Ret.a, and Dex. The combination of 25 mM glucose and 100 nM insulin increased the glycogen content 3.5 times from 50 ± 4 to 179 ± 17 nmol glucosyl U/dish (n = 2). The glycogen content increased to 107 ± 10 nmol glucosyl U/dish (n = 2) in the presence of 25 mM glucose and 10 nM insulin. No other combination of glucose and insulin affected the glycogen accumulation measured at 24 h.

Subsequent experiments on glycogen metabolism were carried out with 5 nM IGF-I, 100 nM Ret.a, and 100 nM insulin on 10- and 14-day-old myotubes. Under these conditions the glycogen content in 10-day-old myotubes increased from basal level at 51 ± 4 at time 0 to 369 ± 36 nmol glucosyl U/mg protein at 6 h (Fig. 2A). Addition of 5 nM Dex increased glycogen accumulation to 606 ± 46 nmol glucosyl U/mg protein at 6 h, corresponding to a 67% increase. This level of glycogen accumulation remained constant until 24 h in the absence as well as in the presence of Dex. Addition of 10 mM Cr had only a small effect on glycogen accumulation for the first 6 h, 25 and 17% increases in the absence and presence of Dex, respectively. From 6 to 24 h, the glycogen level declined to 55 and 62% of the 6-h values in the absence and presence of Dex, respectively.


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Fig. 2.   Glycogen accumulation in 10-day-old (A) and 14-day-old (B) myotubes. Values are means ± SE; n = 4. Cr, creatine; Dex, dexamethasone; AraC, cytosine 1-beta -D-arabinofuranoside; Ret.a, retinoic acid. Significant differences between values (by ANOVA and Tukey's procedure) are marked with identical superscript letters. A: a and b, P < 0.05; c and e, P < 0.01 at 3 and 6 h; d, P < 0.05 at 3, 6, and 24 h. B: a, P < 0.01; b, P < 0.05. Differentiation and culture conditions were as follows for cultures analyzed at day 10 and day 14. Days 0-7, DMEM, 5.05 mM glucose + 10% FCS. For cultures analyzed at day 10: days 7-10, DMEM and 5.05 mM glucose + 2% FCS + 10 µM AraC + 100 nM Ret.a + 5 nM IGF-I ± 5 nM Dex ± 10 mM Cr. For cultures analyzed at day 14: days 7-10, DMEM and 5.05 mM glucose + 2% FCS + 10 µM AraC + 100 nM Ret.a + 5 nM IGF-I ± 10 mM Cr; days 10-11, identical to days 7-10 but without AraC; days 11-14, DMEM and 5.05 mM glucose + 2% FCS + 100 nM Ret.a + 5 nM IGF-I ± 5 nM Dex ± 10 mM Cr. Glycogen accumulation conditions, for both day 10 and day 14, were DMEM and 25 mM glucose + 2% FCS + 100 nM Ret.a + 5 nM IGF-I ± 5 nM Dex ± 10 mM Cr + 100 nM insulin. , without both Cr and Dex; , with Cr but without Dex; open circle , without Cr but with Dex; bullet , with both Cr + Dex. The indicated Cr and Dex concentrations were present during both differentiation and glycogen accumulation periods.

In the 14-day-old myotubes (Fig. 2B), the basal level of glycogen was 48 ± 6 nmol glucosyl U/mg protein. Glycogen accumulation in these cells reached a maximum of 459-555 nmol glucosyl U/mg protein, unaffected by Dex and Cr addition after the first 6 h. In the presence of Dex, independent of Cr addition, the glycogen levels were almost constant from 6 to 24 h (95% of the 6-h values). When Dex was absent, the glycogen level declined to 60% of the 6-h values.

Glucose transport was measured as 3-O-methylglucose uptake in 11-day-old cultures. On day 10, the medium was changed to DMEM without IGF-I but with 5 nM Dex, 100 nM Ret.a, and 10 mM Cr added as we have described (Fig. 2). On day 11, the medium was changed to DMEM, 25 mM glucose + 2% FCS + 100 nM Ret.a + 10 mM Cr with or without 5 nM IGF-I and with or without 100 nM insulin. At 2 h after this medium change, 3-O-methylglucose uptake was (in pmol · mg protein-1 · min-1): 45.2 ± 8.5 in the absence of both insulin and IGF-I, 64.1 ± 4.9 in the presence of 5 nM IGF-I, 70.6 ± 5.8 in the presence of 100 nM insulin, and 78.3 ± 2.8 in the presence of both insulin and IGF-I (n = 4). Thus glucose uptake was significantly stimulated by insulin (P < 0.05) and by the combination of insulin and IGF-I (P < 0.01) after induction of glycogen accumulation.

GS and GPh Activity, G-6-P, and Glucosei During Glycogen Accumulation

Glycogen accumulation was studied in detail in 10-day-old myotubes differentiated with 5 nM IGF-I + 100 nM Ret.a + 5 nM Dex + 10 mM Cr. Glycogen accumulation was initiated with a medium including the same differentation factors and 25 mM glucose + 100 nM insulin. This combination was chosen because it represented the best compromise with respect to PCr/ATP, specific activity of CK, and level of glycogen accumulated.

Glycogen accumulated at a rather constant rate of 315-349 nmol glucosyl U · mg protein-1 · h-1 for the first 2 h (Table 3 and Fig. 3). From 2 to 4 h, the accumulation rate decreased gradually to zero (-22 ± 27 nmol glucosyl U · mg protein-1 · h-1). The maximum level of glycogen, 982 ± 26 nmol glucosyl U/mg, was reached at 4 h, and at 6 h the value was 798 ± 35 nmol glucosyl U/mg (Table 3).

                              
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Table 3.   Glycogen synthase and phosphorylase activities, G-6-P, and intracellular glucose content during glycogen accumulation in 10-day-old L6 myotubes


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Fig. 3.   Glycogen accumulation and percent active a form of glycogen synthase (GS) and phosphorylase (GPh). Values are means ± SE; n = 4. Data were calculated from Table 3. , Glycogen accumulation (cubic spline fit for mean glycogen values for all 4 experiments); bullet , GSa; black-triangle, GPha.

GS and GPh activity. The total activity (a form + b form) of GS and GPh was constant during the accumulation period. The a form of GS was constant from 0 to 0.5 h (30.1 ± 0.9 and 34.7 ± 2.5% of total activity, respectively), but from 0.5 h the activity declined in an exponential manner to 2.8 ± 0.1% at 6 h. This progressive decline in GSa activity was not observed under conditions in which the medium glucose concentration was 0 mM, and no glycogen accumulation was observed (results not shown). The a form of GPh declined from 87 to 75% of the total activity from 0 to 0.5 h (P < 0.05). From 0.5 h, the a form increased to 98% of total activity at 6 h. Although the ratio of GSa to GPha (GSa/GPha) decreased 6.2-fold during the time from 0.5 to 2 h, the glycogen accumulation rate was constant (Fig. 4). Between 2 and 4 h, the glycogen accumulation rate decreased rapidly to zero and GSa/GPha a further 2.5-fold. From 4 to 6 h the glycogen accumulation rate became negative, whereas GSa/GPha decreased only 1.1-fold.


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Fig. 4.   Rate of glycogen accumulation as a function of the ratio of GSa to GPha (GSa/GPha). Values are means ± SE; n = 4. Time points are in parentheses. Rate of glycogen accumulation (expressed as Delta glycogen/Delta t) is the average slope of cubic spline fit in 4 independent experiments at time points indicated in Table 3 and Fig. 3.

G-6-P and glucosei. At the onset of the glycogen accumulation period, the cell content of G-6-P and glucosei was low at 0.5 ± 0.0 and 5 ± 2 nmol/mg protein, respectively (Table 3). The change from conditioned medium with low glucose concentration (5.05 mM at the start of the 72-h period) to medium with 25 mM glucose + 5 nM Dex + 100 nM insulin caused a 9- to 11-fold increase in glucosei and a 5- to 6-fold increase in G-6-P concentration during the 1st h of glycogen accumulation. From 2 to 6 h, both glucosei and G-6-P increased further to 87-117 and 6.8-8.5 nmol/mg protein, respectively.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Differentiation

The present study demonstrates that it is possible to enhance the differentiation level of L6 myotubes as judged by CK activity, PCr/ATP, and contractility. This was obtained by a combination of the differentiation factors Ret.a, IGF-I, and Cr. The CK activity reached under optimal culture conditions tended to decrease with the number of passages of the cells. After the fifth passage, the maximal CK activity was ~1 U/mg protein compared with 10 U/mg protein with the third passage, which was used as a standard in the present experiments.

The CK activity in rat skeletal muscle is 15-35 U/mg protein, depending on the fiber type composition of the muscle (57). Reported values in L6 cells and primary cultures of rat skeletal muscle cells are 0.3-1 and 1-4 U/mg protein, respectively (7, 10, 19, 44), depending on the culture conditions. The highest CK activity obtained in the present experiments was ~9.8 U/mg protein.

IGF-I. The effect of IGF-I on the myogenic differentiation level in cultured cells is in accordance with previously reported results (7-9, 47). Retinoic acid is known to enhance differentiation of many cell types, including myogenic cells (16). In the present study, Ret.a alone had no significant effect on the CK activity. The combination of IGF-I and Ret.a did not affect the CK activity compared with the condition in which only IGF-I was added; however, the myotubes were morphologically more homogeneous than with IGF-I alone. Dex has been reported to increase the myogenic differentiation level in C2C12 and L6 cells, probably by increasing the expression of the IGF-I receptor (55). However, in our experiments, Dex actually had a negative effect as judged by the CK activity (Table 1) and the morphology of the cells.

PCr/ATP. Cr was added to the medium to increase the PCr content and thereby PCr/ATP. The presence of Cr increased the ratio to the levels of skeletal muscles in vivo and did in fact also promote the differentiation level, as reflected in the CK activity. The effect of Cr on PCr/ATP was observed only at a very high concentration of Cr (10 mM), which could indicate that the ability to concentrate Cr, normally displayed by muscle cells (32), is lost in L6 cells. This would seem to contradict the findings of Loike et al. (27), who concluded that Cr transport in L6 cells is an active process in which the Cr transporter, however, is gradually downregulated at a Cr concentration >1 µM.

Glycogen Metabolism

Glycogen accumulation. Reported glycogen levels in rat skeletal muscle are maximally ~850 nmol/mg protein (36). In our experiments, ~50 nmol/mg protein accumulated at basal conditions, and ~1,000 nmol/mg protein accumulated with 25 mM glucose, insulin, and Dex (Table 3). Reported levels of glycogen in L6 cells (in nmol/mg protein with the glucose concentration of medium shown in parentheses) are 22.5 (7 mM) by Pardridge et al. (37), 62 (9 mM) by Huang and Tao (18), and 250 (25 mM) by Wahrmann et al. (54). The low levels of glycogen accumulation in L6 myotubes demonstrated by Pardridge et al. and Huang and Tao may be explained by low intracellular G-6-P levels in response to a low initial extracellular glucose concentration (7 and 9 mM) without insulin or IGF-I addition. This condition will cause low glucose transport, even though the authors demonstrate a high ratio of intracellular glucose to G-6-P. The relatively high glycogen level reported by Wahrmann et al. of 250 nmol/mg protein is at approximately the same level as that observed in the present study, when no differentiation effectors were added. Wahrmann et al. also used a glucose concentration of 25 mM, but no insulin or IGF-I. This suggests that, in their subclone, glucose transport by the GLUT-1 transporter was sufficient to cause glycogen accumulation.

The high glycogen level reached in our experiments is probably a result of increased GS activity (2, 5) and GLUT-4 expression (15, 30, 48) due to the addition of IGF-I, insulin, Ret.a, and Dex combined with increased glucose transport in the presence of a high glucose concentration (25 mM) (25, 30, 33).

The expression of CK and the ability to accumulate glycogen were not correlated when differentiation conditions were improved. Thus the highest CK activities were measured in 14-day-old myotubes, when IGF-I, retinoic acid, and creatine were present, whereas the highest level of glycogen accumulation was measured in 10-day-old myotubes in the presence of IGF-I, Ret.a, Dex, and Cr.

Ten-day-old and 14-day-old cultures exhibited some unexplained differences with respect to the effect of Dex and Cr on glycogen accumulation, suggesting that the regulation of glycogen metabolism was changing with the age of the myotubes. Thus Dex enhanced the accumulation of glycogen in 10-day-old myotubes but appeared to be without effect in 14-day-old myotubes. In 10-day-old myotubes, the glycogen level was constant from 6 to 24 h whether Dex was present or not, whereas Dex had to be present to avoid a fall in the glycogen level after 6 h in 14-day-old myotubes (Fig. 2, A and B). Cr caused a declining glycogen level from 6 to 24 h in 10-day-old myotubes but was without effect in 14-day-old myotubes (Fig. 2, A and B).

GS and GPh. The total activity of GS and GPh was ~15 and 40 mU/mg protein, respectively (Table 3) compared with ~20 and 100-500 mU/mg protein in rat skeletal muscle [GPh measured in the direction of glycogen synthesis and depending on the fiber type composition of the muscle (34, 35)]. The total GS activity was correlated to the CK activity, which was also observed by Wahrmann et al. (54). The total GPh activity was almost constant from day 7 (confluent myoblasts) to day 14 and did not depend on the differentiation level, in contrast to the results reported by Wahrmann et al.

In our experiments, the a form constituted 30.1% of the total GS and 86.4% of the total GPh at the onset of the glycogen accumulation period. The GSa activity did not increase further when glycogen accumulation was induced by insulin, as described by Lazar et al. (26) and Ragolia and Begum (42), presumably because the GS was already activated by inhibition of glycogen synthase kinase-3 by IGF-I, mediated by protein kinase B (2, 5). The level of enzyme phosphorylation in the present study is in agreement with values observed by Wahrmann et al. (54) in L6 myotubes cultured for 9 days with 25 mM glucose (25% for GS and 85% for GPh). These authors also demonstrated that the phosphorylation state changed from 85 to 40% and 25 to 60% for GPh and GS, respectively, by a change in the glucose concentration from 25 to 2.5 mM, thereby decreasing the glycogen level from 125 to 42 nmol/mg protein in 24 h. Huang and Tao (18) found 0.5% GSa and ~80% GPha in 9-day-old myotubes cultured with 9 mM glucose. They observed no change in the phosphorylation state of GS and GPh during 48 h of glycogen accumulation from zero to 62 nmol/mg protein, and they showed that the phosphoprotein phosphatase activity was continuously suppressed by inhibitors.

GSa activity decreased with time concomitant with accumulation of glycogen (Fig. 3) but was constant with time if the rate of glycogen accumulation was minimized by using 0 instead of 25 mM glucose (results not shown), suggesting that the phosphorylation state of GS is subject to feedback regulation by glycogen.

Regulation of the glycogen content. In textbooks of biochemistry, it is often stated that the processes of glycogen synthesis and degradation are mutually exclusive because of concomitant phosphorylation/dephosphorylation of the GS and GPh enzymes (52). However, phosphorylation and dephosphorylation are clearly not the sole regulatory factors for glycogen accumulation in intact skeletal muscles, as pointed out by Chasiotis (4) and Shulman and co-workers (45, 46), and in L6 cells, as shown by the following calculations. At the start of the glycogen accumulation period, the GSa and the GPha activities corresponded to 270 and 1,000 nmol glucosyl U · mg protein-1 · h-1, respectively [when a 2.5-fold lower GPh activity is assumed when assayed in the direction of glycogen degradation compared with the direction of glycogen synthesis (22)]. These figures would seem to exclude any glycogen accumulation at all. Furthermore, the GSa activity decreased, and GPha activity increased during the first 2 h of glycogen accumulation (Fig. 3) despite the fact that the rate of accumulation was constant (Fig. 4). Finally, the fall in GSa with increasing glycogen levels (Fig. 3) indicates lack of correlation between glycogen accumulation and the dephosphorylated form of GS. Consequently, it would seem that regulatory mechanisms other than phosphorylation/dephosphorylation of GS and GPh are playing a major role in L6 cells. These factors might be G-6-P and glucosei. The concentration of G-6-P in the cells was 0.5-1.6 mM, when a protein content of 0.11 mg/mg wet weight and a water content of 60% are assumed (18) (Table 3). Those concentrations would cause a 16-39% activation of GSb and partially phosphorylated forms of GS (calculated from Ref. 18) and could explain the high glycogen accumulation rate. The glucosei concentration was 8-22 mM during the glycogen accumulation period (Table 3 and the same assumptions as for the calculations of G-6-P concentration). Thus GPha could be inhibited by these high concentrations of glucosei corresponding to 5-13 times the inhibitory constant (20). That GPha is in fact inhibited in L6 cells is corroborated by the observation that the initial rate of glycogen degradation in the presence of epinephrine and in the absence of glucose is <10% of the GPha activity (results not shown). The much lower rate of glycogen degradation than the actual GPha activity might, of course, also reflect compartmentation of the enzyme and/or its substrate glycogen and/or rate-limiting activity of the debranching enzyme.

The factor limiting the maximal amount of glycogen accumulated in the incubation period could be the number of available glycogenin/proglycogen molecules, which will limit the number of beta -glycogen molecules that can be synthesized (3, 45). The shape of the curve in Fig. 4 indicates that this could in fact be the case.

    ACKNOWLEDGEMENTS

The expert technical assistance provided by Lis Frandsen, Susanne Johannessen, Ida Tønnesen, Hanne Wennicke, and Peter Vingaard-Larsen is highly appreciated.

    FOOTNOTES

Financial support was obtained from Danish Medical Research Council, Beckett Fonden, Savværksejer Jeppe Juhl og hustru Ovita Juhls Mindelegat, the Danish National Research Council, and The Novo Nordic Foundation.

Address for reprint requests: P. Elsner, IMBG, Biochemistry Laboratory A, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.

Received 22 December 1997; accepted in final form 13 August 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Albagli-Curiel, O., G. Carnac, M. Vandromme, S. Vincent, P. Crépieux, and A. Bonnieu. Serum-induced inhibition of myogenesis is differentially relieved by retinoic acid and triiodothyronine in C2 murine muscle cells. Differentiation 52: 201-210, 1993[Medline].

2.   Alessi, D. R., M. Andjelkovic, B. Caudwell, P. Cron, N. Morrice, P. Cohen, and B. A. Hemmings. Mechanism of activation of protein kinase B by insulin and IGF-I. EMBO J. 15: 6541-6551, 1996[Abstract].

3.   Alonso, M., J. Lomako, W. M. Lomako, and W. J. Whelan. A new look at the biogenesis of glycogen. FASEB J. 9: 1126-1137, 1995[Abstract/Free Full Text].

4.   Chasiotis, D. The regulation of glycogen phosphorylase and glycogen breakdown in human skeletal muscle. Acta Physiol. Scand. Suppl. 518: 1-68, 1983.

5.   Cross, D. A., D. R. Alessi, P. Cohen, M. Andjelkovic, and B. A. Hemmings. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378: 785-789, 1995[Medline].

6.   El Abida, K., A. Duvallet, L. Thieulart, M. Rieu, and M. Beaudry. Lactate transport during differentiation of skeletal muscle cells: evidence for a specific carrier in L6 myotubes. Acta Physiol. Scand. 144: 469-471, 1992[Medline].

7.   Ewton, D. Z., S. L. Roof, K. A. Magri, F. J. McWade, and J. R. Florini. IGF-II is more active than IGF-I in stimulating L6A1 myogenesis: greater mitogenic actions of IGF-I delay differentiation. J. Cell. Physiol. 16: 277-284, 1994.

8.   Florini, J. R., D. Z. Ewton, and K. A. Magri. Hormones, growth factors and myogenic differentiation. Annu. Rev. Physiol. 53: 201-216, 1991[Medline].

9.   Florini, J. R., and K. A. Magri. Effects of growth factors on myogenic differentiation. Am. J. Physiol. 256 (Cell Physiol. 25): C701-C711, 1989[Abstract/Free Full Text].

10.   Funanage, V. L., S. S. Smith, and M. A. Minnich. Entactin promotes adhesion and long-term maintenance of cultured regenerated skeletal myotubes. J. Cell. Physiol. 150: 251-257, 1992[Medline].

11.   Gabbert, H. E., C.-D. Gerharz, H.-K. Biesalski, R. Engers, and C. Luley. Terminal differentiation and growth inhibition of a rat rhabdomyosarcoma cell line (BA-HAN-1C) in vitro after exposure to retinoic acid. Cancer Res. 48: 5264-5269, 1988[Abstract].

12.   Gilboe, D. P., K. L. Larson, and F. Q. Nuttall. Radioactive method for the assay of glycogen phosphorylases. Anal. Biochem. 47: 20-27, 1972[Medline].

13.   Golden, S., P. A. Wals, and J. Katz. An improved procedure for the assay of glycogen synthase. Anal. Biochem. 77: 436-445, 1977[Medline].

14.   Gomori, G. Ciba-Geigy Scientific Tables (7th ed.). Basel: Ciba-Geigy, 1970, p. 280.

15.   Guillet-Deniau, I., A. Leturque, and J. Girard. Expression and cellular localization of glucose transporters (GLUT-1, GLUT-3 and GLUT-4) during differentiation of myogenic cells isolated from rat foetuses. J. Cell. Sci. 107: 487-496, 1994[Abstract/Free Full Text].

16.   Halevy, O., and O. Lerman. Retinoic acid induces adult muscle cell differentiation mediated by the retinoic acid receptor-alpha . J. Cell. Physiol. 154: 566-572, 1993[Medline].

17.   Hespel, P., and E. A. Richter. Glucose uptake and transport in contracting, perfused rat muscle with different pre-contracting glycogen concentrations. J. Physiol. (Lond.) 427: 347-359, 1990[Abstract].

18.   Huang, F. L., and S.-H. Tao. Glycogen metabolism in myogenic cells in culture. Presence of inhibitors for dephosphorylation of glycogen synthase and glycogen phosphorylase. Arch. Biochem. Biophys. 199: 123-132, 1980[Medline].

19.   Jacobs, A. E. M., A. A. G. M. Benders, A. Oosterhof, and J. H. Veerkamp. Effects of growth medium, electrical stimulation and paralysis on various enzyme activities in cultured rat muscle cells. Comparison with activities in rat muscles in vivo. Int. J. Biochem. 24: 751-758, 1992[Medline].

20.   Kasvinsky, P. J., R. J. Fletterick, and N. B. Madsen. Regulation of the dephosphorylation of glycogen phosphorylase a and glycogen synthase b by glucose and caffeine in isolated hepatocytes. Can. J. Biochem. 59: 387-395, 1981[Medline].

21.   Katz, J., S. Golden, and P. A. Wals. Stimulation of hepatic glycogen synthesis by amino acids. Proc. Natl. Acad. Sci. USA 73: 3433-3437, 1976[Abstract].

22.   Katz, J., S. Golden, and P. A. Wals. Glycogen synthesis by rat hepatocytes. Biochem. J. 180: 389-402, 1979[Medline].

23.   Kidokoro, Y. Developmental changes of membrane electrical properties in a rat skeletal muscle cell line. J. Physiol. (Lond.) 244: 129-143, 1975[Abstract].

24.   Kissane, J. M., and E. Robins. The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the nervous system. J. Biol. Chem. 233: 184-188, 1958[Free Full Text].

25.   Klip, A., G. Li, and W. J. Logan. Induction of sugar uptake response to insulin by serum depletion in fusing L6 myoblasts. Am. J. Physiol. 247 (Endocrinol. Metab. 10): E291-E296, 1984[Abstract/Free Full Text].

26.   Lazar, D. F., R. J. Wiese, M. J. Brady, C. C. Mastick, S. B. Waters, K. Yamauchi, J. E. Pessin, P. Cuatrecasas, and A. R. Saltiel. Mitogen-activated protein kinase inhibition does not block the stimulation of glucose utilizatioin by insulin. J. Biol. Chem. 270: 20801-20807, 1995[Abstract/Free Full Text].

27.   Loike, J. D., D. L. Zalutsky, E. Kaback, A. F. Miranda, and S. C. Silverstein. Extracellular creatine regulates the creatine transport in rat and human muscle cells. Proc. Natl. Acad. Sci. USA 85: 807-811, 1988[Abstract].

28.   Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-276, 1951[Free Full Text].

29.   Luzatti, D., and G. Drugeon. The effect of actinomycin D on RNA and protein synthesis during differentiation of myoblasts of line L6E: the half-life of functional myosin messenger. Biochimie 54: 1157-1167, 1972[Medline].

30.   Maher, F., S. Clark, and L. C. Harrison. Chronic stimulation of glucose transporter gene expression in L6 myocytes mediated via the insulin-like growth factor-1 receptor. Mol. Endocrinol. 3: 2128-2135, 1989[Abstract].

31.   Marchal, S., I. Cassar-Malek, F. Pons, C. Wrutniak, and G. Cabello. Triiodothyrosine influences quail myoblast proliferation and differentiation. Biol. Cell 78: 191-197, 1993[Medline].

32.   Meyer, R. A. Linear dependence of muscle phosphocreatine kinetics on total creatine content. Am. J. Physiol. 257 (Cell Physiol. 26): C1149-C1157, 1989[Abstract/Free Full Text].

33.   Mirsumoto, Y., E. Burdett, A. Grant, and A. Klip. Differential expression of the GLUT1 and GLUT4 glucose transporters during differentiation of L6 muscle cells. Biochem. Biophys. Res. Commun. 175: 652-659, 1991[Medline].

34.   Moruzzi, E. V., T. L. Cubeddu, and E. Bergamini. Developmental changes of glycogen enzymes in fast and slow muscles of the rat. Growth 43: 73-79, 1979[Medline].

35.   Moruzzi, E. V., T. L. Cubeddu, and E. Bergamini. Adrenaline and glycogen synthase activity in fast and slow muscles of rat. FEBS Lett. 111: 361-364, 1980[Medline].

36.   Nakatani, A., D.-H. Han, P. A. Hansen, L. A. Nolte, H. H. Host, R. C. Hickner, and J. O. Holloszy. Effect of endurance exercise training on muscle glycogen supercompensation in rats. J. Appl. Physiol. 82: 711-715, 1997[Abstract/Free Full Text].

37.   Pardridge, W. M., M. B. Davidson, and D. Casanello-Ertl. Glucose and amino acid metabolism in an established line of skeletal muscle cells. J. Cell. Physiol. 96: 309-318, 1978[Medline].

38.   Passonneau, J. V., and O. H. Lowry. A collection of metabolite assays. In: Enzymatic Analysis. Totowa, NJ: Humana, 1993, chapt. 6, p. 121-122.

39.   Passonneau, J. V., and O. H. Lowry. A collection of metabolic assays. In: Enzymatic Analysis. Totowa, NJ: Humana, 1993, chapt. 6, p. 162-163.

40.   Passonneau, J. V., and O. H. Lowry. A collection of enzyme assays. In: Enzymatic Analysis. Totowa, NJ: Humana, 1993, chapt. 7, p. 246-248.

41.   Patrick, J., S. F. Heinemann, J. Lindstrom, D. Schubert, and J. H. Steinbach. Appearance of acetylcholine receptors during differentiation of a myogenic cell line. Proc. Natl. Acad. Sci. USA 69: 2762-2766, 1972[Abstract].

42.   Ragolia, L., and N. Begum. The effect of modulating the glycogen-associated regulatory subunit of protein phospharase-1 on insulin action in rat skeletal muscle cells. Endocrinology 138: 2398-2404, 1997[Abstract/Free Full Text].

43.   Rösler, K. M., V. Askanas, W. K. Engel, and A. Martinuzzi. Effects of electrical stimulation and tetrodotoxin paralysis on expression of muscle-specific isoenzymes of four enzymes in aneurally cultured embryonic rat muscle. Exp. Neurol. 97: 739-745, 1987[Medline].

44.   Shainberg, A., G. Yagil, and D. Yaffe. Alterations of enzymatic activities during muscle differentiation in vitro. Dev. Biol. 25: 1-29, 1971[Medline].

45.   Shulman, R. G., G. Bloch, and D. L. Rothman. In vivo regulation of muscle glycogen synthase and the control of glycogen synthesis. Proc. Natl. Acad. Sci. USA 92: 8535-8542, 1995[Abstract].

46.   Shulman, R. G., and D. L. Rothman. Enzymatic phosphorylation of muscle glycogen synthase: a mechanism for maintenance of metabolic homeostasis. Proc. Natl. Acad. Sci. USA 93: 7491-7495, 1996[Abstract/Free Full Text].

47.   Silverman, L. A., Z.-Q. Cheng, D. Hsiao, and S. M. Rosenthal. Skeletal muscle cell-derived insulin-like growth factor (IGF) binding proteins inhibit IGF-I-induced myogenesis in rat L6E9 cells. Endocrinology 136: 720-726, 1995[Abstract].

48.   Sleeman, M. W., H. Zhou, S. Rogers, K. W. Ng, and J. D. Best. Retinoic acid stimulates glucose transporter expression in L6 muscle cells. Mol. Cell. Endocrinol. 108: 161-167, 1995[Medline].

49.   Sokolov, I. N., S. N. Lyzlova, and I. P. Askanarin. Creatine induction of creatine kinase synthesis in a developing monolayer culture of muscle cells. Biokhimiia 40: 3-7, 1975[Medline].

50.   Steinbach, J. H. Acetylcholine responses on clonal myogenic cells in vitro. J. Physiol. (Lond.) 247: 393-405, 1975[Abstract].

51.   Steinbach, J. H., A. J. Harris, J. Patrick, D. Schubert, and S. Heinemann. Nerve-muscle interaction in vitro. Role of acetylcholine. J. Gen. Physiol. 62: 255-270, 1973[Abstract/Free Full Text].

52.   Stryer, L. Glycogen metabolism. In: Biochemistry. New York: Freeman, 1988, chapt. 19, p. 462-463.

53.   Turo, K. A., and J. R. Florini. Hormonal stimulation of myoblast differentiation in the absence of DNA synthesis. Am. J. Physiol. 243 (Cell Physiol. 12): C278-C284, 1982[Abstract].

54.   Wahrmann, J. P., F. Gros, and D. Luzzati. Phosphorylase and glycogen synthase during myoblast differentiation. Biochimie 55: 457-463, 1973[Medline].

55.   Whitson, P. A., C. A. Stuart, H. Huls, C. F. Sams, and N. M. Cintron. Dexamethasone effects on creatine kinase activity and insulin-like growth factor receptors in cultured muscle cells. J. Cell. Physiol. 140: 8-17, 1989[Medline].

56.   Yaffe, D. Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc. Natl. Acad. Sci. USA 61: 477-489, 1968[Medline].

57.   Zuurveld, J. G. E. M., P. Wirtz, H. M. T. Loermans, and J. H. Veerkamp. Postnatal growth and differentiation in three hindlimb muscles of the rat. Cell Tissue Res. 241: 183-192, 1985[Medline].


Am J Physiol Endocrinol Metab 275(6):E925-E933
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