Glycogen metabolism in myogenic cells in culture. Presence of inhibitors for dephosphorylation of glycogen synthase and glycogen phosphorylase

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 199, No. 1, January, pp. 123-132, 1980

Glycogen Metabolism for Dephosphorylation

in Myogenic of Glycogen

FREESIA

Cells in Culture. Presence of Inhibitors Synthase and Glycogen Phosphorylase

L. HUANG

AND

SHYY-HWA TAO

Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, and Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 Received

May 23, 1979; revised

August 15, 1979

Myoblasts of L6 cells proliferate and fuse normally into myotubes in culture. In cell extracts of growing or confluent cultures, glycogen phosphorylase is predominantly in the a form and glycogen synthase is in the D form. Despite the predominant presence of D-form synthase and a-form phosphorylase, glycogen content reaches 12 pgimg protein in confluent cultures 4 to 6 h after feeding of fresh medium. Both D-form synthase and a-form phosphorylase in the cell extract cannot be converted into their corresponding I form and b form enzymes upon incubation at 37°C. However, the enzymes can be converted into their corresponding dephospho-forms by added rabbit muscle phosphoprotein phosphatase. Measurement of the phosphoprotein phosphate activity with rabbit muscle [32P]phosphorylase a as substrate reveals that the specific activity of the enzyme increases with increasing dilution of the cell extract. In addition, synthase and phosphorylase recovered from high-speed centrifugation can be converted into their corresponding dephospho-forms of enzyme by incubation with MnCl,. These conversions can be reversed by the addition of ATP, Mg*+, and cyclic AMP and they are inhibited by the high-speed supernatant fraction which also inhibits the purified rabbit muscle phosphoprotein phosphatase. It is concluded that lack of conversion of glycogen synthase and phosphorylase in crude extract of L6 cells is due to the presence of phosphatase inhibitor. Inhibitor activity in the high-speed supernatant fluid is heat stable, nondialyzable, and trypsin sensitive. Two main inhibitor peaks are resolved by chromatography on a DEAE-cellulose column. Activity of the first peak from the column is relatively unaffected by protein kinase treatment, while that of the second peak is increased by protein kinase.

L6 cells are differentiating myoblast cultures of rat origin (1). They exhibit many characteristic features of in uivo muscle differentiation. Mononucleated myoblasts grow exponentially until reaching confluency and then begin to fuse into multinucleated myotubes (1). Accumulation of the characteristic muscle proteins, including myosin, actin, troponin, and tropomyosin, and increase in the specific activities of glycogen phosphorylase, creatine kinase, and glycogen synthase, accompany the fusion process (2-4). Membrane-bound adenylate cyclase is known to be present in L6 cells (3, 5), and low levels of adenylate cyclase and cyclic AMP are found to associate with cell fusion or myoblast differentiation (3, 5-7). Whether the adenylate cyclase is hormone responsive is not known, but insulin has 123

been reported to stimulate myogenesis of the same myoblast line (8). Regulation of glycogen metabolism has been studied in various mammalian tissues, but little was known about the regulatory properties of the cultured cells derived from these tissues. In cultured brain cells of astrocytoma and neuroblastoma (9), both glycogen synthase and phosphorylase vary with the concentration of glucose in the medium. These cells behave similarly to that of perfused rat liver (lo), in which a high level of glucose causes a rapid inactivation of phosphorylase and a conversion of D- to I-form glycogen synthase. On the contrary, cultured choriocarcinoma cells (11) seem to have a different regulatory mechanism for glycogen metabolism. In these cells, a high concentration of glucose 0003.9861/80/010123-10$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved

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in medium does not result in the conversion of D- to I-form synthase or a- to b-form phosphorylase and there is continued glycogen synthesis in the presence of D-form glycogen synthase. In rabbit skeletal muscle, we recently provided evidence (12-14) that at least two heat-stable protein inhibitors are involved in the regulation of phosphoprotein phosphatase, the enzyme responsible for the conversion of D- to I-form synthase and ato b-form phosphorylase. Khandelwal et al. (15) reported that in streptozotocin-induced diabetic rat liver, the increase in glycogen synthase I activity following insulin treatment was associated with a concomitant increase in phosphoprotein phosphatase activity and a decrease in inhibitor activity. Our studies with an isolated hindlimb perfusion system showed that adrenaline administration rapidly decreased muscle phosphorylase phosphatase activity and increased heat-stable phosphatase inhibitor activity (16). This was associated with increased tissue cyclic AMP concentration, phosphorylase activation, and glycogen synthase inactivation. However, a step(s) leading to alteration of levels of inhibitor in the hormonal regulation of phosphoprotein phosphatase was not known. We wished to learn the regulatory properties of glycogen metabolism in cultured muscle cells so that such cultures could be used for studying the action of inhibitors in hormonal regulation. Cell cultures are more convenient than intact animals or organ perfusion technics for this purpose. MATERIALS

AND METHODS

UDP-glucose, glucose-6-P,’ glucose-l-P, rabbit liver glycogen, and phosphorylases a and b were obtained from Sigma Chemical Company (St. Louis, MO.). UDP[14C]glucose, [“‘Clglucose-l-P, and [Y-~*P]ATP were from New England Nuclear Corporation (Boston, Mass.). Ham’s F12 medium and fetal bovine serum were from Flow Laboratories (Rockville, Md.).

’ Abbreviations used: -P, phosphoric residue; DTT, dithiothreitol; Buffer A, 50 mM Tris-Cl, pH 7.4, containing 5 mM EDTA, 1 mM DTT, 0.05 mM phenylmethylsulfonylfluoride, and 50 mM KF; Buffer B, 50 mM Tris-Cl, pH 7.4, containing 2 mM DTT.

AND TAO Glycogen synthase activity was determined by following the incorporation of glucose from UDP-[Y]glucose into glycogen. The 25-~1 reaction mixture contained 40 mM Tris-Cl buffer, pH 7.8, 8 mg/ml of rabbit liver glycogen, 4 mM UDP-glucose (2.5 x IO5 cpm), 10 mM glucose-6-P or Na$O, and enzyme. Separation of the resulting [“Clglycogen from UDP[“Qglucose was achieved by chromatography as described elsewhere (17). One unit of activity is defined as that amount of enzyme which catalyzed the incorporation of 1 pmol of glucose from UDP-glucose/ min into glycogen at 37°C. Percentage of synthase I is defined as the activity without glucose-6-P divided by the activity with glucose-6-P multiplied by 100. Glycogen phosphorylase activity was determined by following the incorporation of glucose from [“‘Clglucose-l-P into glycogen. The 25-~1 reaction mixture contained 20 mM ,&glycerophosphate buffer, pH 6.8; 10 mg/ml of rabbit liver glycogen; 1 mM AMP; 16 mM glucose-l-P (2.5 x lo5 cpm) and enzyme. Separation of [Ylglycogen from [‘4C]glucose-l-P was carried out as that for the assay of glycogen synthase. One unit of activity is defined as that amount of enzyme which catalyzes the incorporation of 1 pmol of glucose from glucose-1-Pimin into glycogen at 37°C. Phosphorylase phosphatase activity was measured by the release of 32P from [9’P]phosphorylase a. [32P]Phosphorylase a was prepared from rabbit muscle phosphorylase b according to Torres and Chelala (18). A unit of activity is defined as that amount of enzyme which releases 1 pmol of [SzP]phosphate/min at 37°C. Inhibitor activity was determined as the percentage inhibition of rabbit muscle phosphorylase phosphatase in an assay which contained 5 FLU of phosphatase. Phosphorylase phosphatase used in the inhibitor assay was prepared as previously described (12). Rabbit muscle phosphoprotein phosphatase used in the enzyme conversion was prepared according to Brandt et al. (19). Rabbit muscle cyclic AMP-dependent protein kinase for the phosphorylation of inhibitor was prepared by the method of Wastila el al. (20) and was further purified by hydroxyapatite column chromatography (21). Phosphorylation of the inhibitors was carried out by incubation of the inhibitors for 10 min at 37°C with 30 mu/ml protein kinase, 0.05 mM cyclic AMP, 0.05 mM ATP, and 5 mM MgCl,. Glucose concentration was determined enzymatically with hexokinase and glucose-6-P dehydrogenase (22). Glycogen was measured with amylo-cu-1,4-a-1,6-glycosidase, hexokinase, and glucose-6-P dehydrogenase (23). Protein concentration was determined by the method of Lowry (24). Cells of myogenic line L6 (1) were grown in 75cm2 plastic flasks in a humidified incubator (95% air, 5% CO,) at 37°C with Ham’s F-12 medium supplemented with 10% fetal bovine serum, streptomycin (100 pgiml), and penicillin (100 unit/ml). The medium was changed

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every 2-3 days. Cells reached confluency on the fifth to sixth day after splitting and were usually harvested on the seventh to eighth day unless otherwise indicated. Cells were collected by scraping off the flask with a rubber policeman after washing twice with buffer of 50 mM Tris-Cl, pH 7.4, containing 5 mM EDTA, 1 mM DTT, 0.05 ITIM phenylmethylsulfonylfluoride, and 50 mM KF (Buffer A). The cell suspension was first sonicated in a Raytheon sonic oscillator (250 W. 10 kc, 2 min) and then centrifuged (7700 9, 10 min). Cell extracts were used at once for the measurement of glycogen synthase and phosphorylase activities. For the measurement of enzyme conversion, cell extracts were dialyzed against buffer of 50 mM Tris-Cl, pH 7.4, containing 2 mM DTT (Buffer B) before testing. High-speed centrifugation (15O,OOOy,60 min) was used to separate particulate and soluble fraction.

RESULTS

L6 cells proliferate rapidly in culture medium during the first 4-5 days after plating and reach confluency at approximately fifth to sixth day. The changes in total cellular protein and in glycogen content are shown in Fig. 1. The specific activity of glycogen phosphorylase increases rapidly during growth; however, such activity in the absence of AMP is approximately 85% of that in the presence of AMP. The specific

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activity of glycogen synthase stays fairly constant during growth, but such activity in the absence of glucose-6-P is very low. Thus, under normal growing conditions, despite the accumulation of glycogen, glycogen synthase is entirely in the D form and phosphorylase is predominantly in the a form. A closer examination of glycogen phosphorylase and synthase activities in confluent L6 cultures reveals that both enzymes are predominantly in the phosphorylated forms over a 48-h period after replenishment of fresh medium (Fig. 2). Intracellular concentrations of glycogen increase during the initial 5 to 6 h of incubation and gradually decline thereafter, even though the concentration of glucose in the medium is still at 8 mM. The rate of glucose consumption in the medium is linear during the 24-h incubation. At the peak concentration of glycogen only 5% of glucose consumed can be accounted for as glycogen. These results indicate that despite the high glucose concentration in the medium, glycogen phosphorylase is in its active a form and glycogen synthase is completely in the inactive D form. Nonetheless, glycogen is accumulated in the presence of D-form glycogen synthase. It appears that the normal increases in

IDAY)

FIG. 1. Glycogen and protein contents and glycogen synthase and phosphorylase

activities of growing I,6 cultures. Confluent cells were trypsinized and inoculated into ‘i5-cm’tissue culture flasks containing 20 ml of medium. The medium was changed every day. At indicated times, which Lvere 4-5 h after changing with fresh medium, two flasks were taken for the measurement of glycogen and protein contents and the enzyme activities in the crude cell extract (four flasks were taken before the fourth day. since mixing of two flasks was necessary to make enough cell mass for measurement). Each point represents the average of the two measurements. The symbols are: protein, n ; glycogen, 0; specific activity of glycogen phosphorylase with, 0, and without, 0, AMP; specific activity of glycogen synthase with, A, and without, A, glucose-6-P.

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FIG. 2. Glucose consumption, glycogen content, and specific activities of synthase and phosphorylase in confluent L6 cultures after replenishment with fresh medium. The cells were grown for 7 days in 75-cm2 flasks and the experiment was started by feeding with 10 ml fresh medium. At indicated times, samples were taken for the various measurements. Each time point represents the average of two measurements. The symbols are: glucose consumed, W; glycogen content, Cl; phosphorylase activity with, 0, and without, 0, AMP; specific activity of synthase with, A, and without,& glucose-6-P.

I-form synthase and decreases in u-form phosphorylase with glycogen synthesis and glucose stimulation of enzyme conversion do not exist in the cultured L6 cells. In crude extracts of confluent L6 cells, glycogen phosphorylase is predominantly in the a form and synthase in the D form. Incubation of the dialyzed extract at 37°C does not result in the conversion of either phosphorylase or synthase into their corresponding b and I forms of the enzyme. Even in the presence of 50 mM glucose, which releases the AMP inhibition of phosphatase, or 2 mM MnCl,, which is a phosphatase activator, specific activity of phosphorylase or synthase remained practically unchanged over a period of 60 min. The conversion mechanism in these extracts seemed to be impaired in all extracts prepared from cells after various lengths of incubation with the fresh medium. Although incubation of the L6 cell extract does not result in the conversion of synthase or

phosphorylase, the conversion can be observed by incubation of the dialyzed extracts at 37°C with purified rabbit muscle phosphoprotein phosphatase (Fig. 3). The conversion does not require Mn2+ and, in fact, is slightly inhibited in its presence. When the dialyzed cell extracts were diluted and assayed against rabbit muscle [32P]phosphorylase a, the specific activity of phosphatase increased at higher dilutions (Fig. 4). This samephenomenon has also been observed in dialyzed rat muscle or rabbit muscle extracts (F. L. Huang, unpublished observation), and was mostly due to the presence of phosphatase inhibitors. The presence of such inhibitors in extracts of L6 cells would explain the lack of conversion of glycogen Synthase and phosphorylase in the concentrated extract. A crude extract of L6 cells was fractionated by high-speed centrifugation at 150,OOOg for 1 h. Both the pellet and supernatant fractions were assayed: more than 85% of total synthase and phosphorylase activities were recovered in the pellet fraction after high-speed centrifugation (Table I). Incubation of the pellet enzymes at 37°C does not result in any conversion of the enzyme (Figs. 5A and B). However, in the presence of 2 mM MnC12, conversions of phosphoryl-

FIG. 3. Conversions of crude extract enzymes by rabbit muscle phosphatase. Dialyzed cell extract of confluent L6 cultures was incubated (4.71 mg/ml) with rabbit muscle phosphoprotein phosphatase (1.3 mu/ml reaction mixture as assayed with [3ZP]phosphorylase a). At indicated times, aliquots were removed for phosphorylase assay with, 0, and without, 0, AMP and for synthase assay with, A, and without, A, glucose-6-P. A and B, extract and phosphatase; C and D, extract, phosphatase and 2 mM MnCl,.

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FIG. 4. Specific activity of phosphorylase phosphatase activity as a function of cell extract concentration. Cell extract with various dilutions were assayed with 32P-labeled rabbit muscle phosphorylase a. Specific activities were plotted against protein content in 50 ~1 of assay mixture.

ase a to b and synthase D to I are observed (Figs. 5C and D). With addition of the supernatant fraction from high-speed centrifugation, this conversion is completely inhibited. So, lack of conversion of glycogen phosphorylase and synthase in L6 cells is probably due to the presence of some inhibitory substances in the supernatant fraction. In addition, the conversion of phosphorylase and synthase in the pellet fraction is reversible by addition of ATP, MgC&, and cyclic AMP (Fig. 6). These observations show that the conversion of the pellet enzymes is a phosphatase reaction and the reversal reaction is catalyzed by a kinase reaction. The inhibitory substance in the supernatant fraction of high-speed centrifugation is therefore phosphatase inhibitor. The supernatant fractions from highspeed centrifugation also inhibit rabbit muscle phosphorylase phosphatase activity when assayed with [“2P]phosphorylase a. The inhibition is concentration dependent (Fig. 7). Calculated from the linear portion

TIME ,Ml”,

FIG. 5. Conversions of the pelleted phosphorylase and synthase in the presence of MnCl, and inhibition of conversion by the supernatant fluid from high-speed centrifugation. Pellet fraction from high-speed centrifugation (150,OOOg 1 h) was washed and resuspended in Buffer B and was incubated (120 ~1 of 2.44 mg/ml) at 37°C. At indicated times, aliquots were removed forphosphorylase assay with, 0, and without, 0, AMP and for synthase assay with, A, and without, A, glucose-6-P. A and B, extract alone; C and D, extract with 2 InM MnCl,; E and F, extract with MnCl, and the supernatant fluid (2.56 mgiml reaction) of high-speed centrifugation which has been dialyzed against Buffer B. Final dilution of the supernatant fluid was one-half of that originally present in the crude extract. Activities were expressed as mU of 120 ~1 reaction mixture.

(O-40%) of the inhibition curve, the activity is 3.6 mU of phosphatase inhibitedimg protein. Activity for the heat-treated rat muscle extract was 3 mU phosphatase inhibited/mg protein (16).

TABLE

I

SUBCELLULARDISTRIBUTIONOFGLYCOGENPHOSPHORYLASEAND SYNTHASEIN I,6 CELLS mu/ml

of crude extract

Phosphorylase Fractions Crude extract High-speed supernatant High-speed pellet

-AMP 145.0 12.6 144.0

Synthase

+AMP 157.0 15.9 162.0

-Glucose-6-P 1.47 0.11 0.92

+Glucose-6-P 34.7 2.9 29.6

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AND TAO TABLE

II

PROPERTIESOFPHOSPHATASEINHIBITORINTHE SUPERNATANTFLUIDOFHIGH-SPEED CENTRIFUGATION Treatment

FIG. 6. Reversal of enzyme conversion. Pellet fractions were incubated in the presence of 2 mM MnCl, for 60 min (Fig. 6). After 2 h of dialysis against Buffer B the reversal reaction was initiated by adding 1 mM ATP, 5 mM MgCl, and 0.05 mM cyclic AMP. Percentage phosphorylase a, 0, 0; percentage synthase I, A, A.

Table II shows the effects of some treatments on the inhibitor activity of the supernatant fluid. Treatment by ribonuclease or alkaline phosphatase does not change the inhibitor activity, but treatment with trypsin reduces the activity substantially. Thus, the inhibitors may be nondialyzable and heat-stable protein molecules. Inhibitors could be separated by chromatography on a DEAE-cellulose column (Fig. 8). A major activity peak was eluted from the column at 0.09 M KCl; minor activity was eluted at 0.17 M KCl. When column fractions were treated with protein kinase, the major activity peak was only slightly affected, whereas the minor activity peak at 0.17 M KC1 was greatly increased.

FIG. 7. Effect of heated high-speed supernatant fluid from L6 cells on rabbit muscle phosphorylase activity. Increasing amounts of dialyzed and heated (lOO”C, 5 min) supernatant fluid were added to phosphatase assay (50 ~1) where 5 FU of rabbit muscle phosphatase was tested with :“P-labeled rabbit muscle phosphorylase a.

None Dialysis” Heating” Trypsin’,” Ribonuclease”,’ Alkaline phosphatased,’

Percentage

inhibition

39.4 38.8 37.5 6.1 42.2 31.5

o Overnight dialysis against Buffer B at 4°C. h Heating was at 100°C for 5 min. ’ Trypsin treatment was 40 pg trypsiniml for 10 min followed by addition of a five-fold excess by weight of soybean trypsin inhibitor. d Buffer B with enzyme but without supernatant fluid was used as control to assure that inhibition was not brought forth by the testing enzyme. Enzyme treatment was at 37°C. p Four hours with 30 fig ribonuclease A/ml. ’ Fifteen minutes with 15 pgalkaline phosphatase/ml.

There was minor activity at the beginning of the gradient. This activity pattern was very different from that of rabbit skeletal muscle (14) in which the kinase-activatable inhibitor was eluted at lower salt concentration from the salt gradient during chromatography than the other type of inhibitor, whose activity was not affected by the kinase. Furthermore, when aged high-speed fluid was separated on the same column, inhibitor activity showed up in many more peaks by salt elution (not illustrated). This could be the result of proteolysis, since freshly prepared and heated supernatant fluid did not give such a result. Since conversion reaction of synthase was not detected and glycogen was synthesized in the presence of D-form synthase, we were interested in an alternative mechanism for the activation of D-form synthase. Effects of various salts on glycogen synthase activity -. were studied. In the absence of glucose-&P, 5 mM MnSo, activates synthase go-fold, and percentage synthase I is 40 (Table III). The activation is due to the presence of the Mn*+ and SO;- since it is also observed in the combination of MnCI, and (NHJ2S04, whereas MnCl, or (NH&SO, alone or other

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FIG. 8. DEAE-cellulose column chromatography of phosphatase inhibitors from high-speed supernatant fluid of L6 cells. Heated and dialyzed high-speed supernatant fluid (19 ml, 9.5 mg protein) was loaded on a DE-52 column (1.5 x 12 cm) which was previously equilibrated with 5 mM Tris-Cl, pH 7.4, containing 0.25 mM EDTA and 0.25 mM DTT. After extensive washing with equilibrating buffer, the column was eluted with KC1 gradient in the same buffer. One hundred fractions of 2 ml were collected. Fractions were assayed for inhibitor activity against rabbit muscle phosphatase before (0) and after (0) protein kinase treatment. Conditions for kinase treatment were incubations for 10 min with 0.05 mM ATP, 5 mM MgCl,, 0.05 mM cyclic AMP, and 30 mu/ml protein kinase. After kinase treatment, fractions were fivefold diluted into phosphatase assay. Equilibration buffer, treated the same as fractions, was tested in control nhosphatase to assure that the inhibition was not due to the carrying - _ over ingredient from the kinase treatment. TABLE

III

EFFECTSOF SALTSON GLYCOGEN SYNTHASEACTIVITY Activityb

Addition” None NH&l MgCl, CaCl, M&l, (NHMO, M@O, CaSO, MnSO, MnCl, + potassium phosphate MnCl, + (NH,),SO,

+ Glucose6-P 6.06 6.00 8.30 5.23 6.15 5.24 6.92 8.84 6.72

(l.O)e (1.0) (1.4) (0.9) (1.0) (0.9) (1.1) (1.5) (1.1)

-Glucose6-P 0.03 0.06 0.03 0.14 0.23 0.02 0.08 0.26 2.75

(1.0)’ (2.0) (1.0) (4.7) (9.3) (0.7) (2.7) (8.7) (91.7)

Percentage I

salt does not induce such an effect. In the presence of glucose-&P, metal sulfate and other salts affect synthase only minimally. The activation-saturation curves for MnSO, in the absence and presence of glucose-6-P are sigmoidal (Fig. 9). The A,,, value for MnSO, in the absence of glucose-6 P is 4.4 mM. In the presence of 1,5, or 10 mM

0.5 1.0 0.4 2.6 4.6 0.4 1.2 2.9 40.9

4.10 (0.7)

0.15 (5.0)

3.6

7.83 (1.3)

2.46 (82.0)

31.4

Mn SO4 ImM)

a Concentration of the various salts was 5 mM. b Activity is expressed as nmol of glucose incorporated into glycogen during 20 min of incubation. c Values in parentheses are -fold increases in activities as compared to control.

FIG. 9. Effect of MnSO, on glycogen synthase activity. Extract of confluent L6 cells was used. Activity is expressed as nmol of glucose incorporated into glycogen during 20 min of incubation. The concentrations of glucose-6-P: 0 mM, 0; 1 mM, 0; 5 mM, A; and 10 mM, n .

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V[GLUCOSE-6-P]

FIG. 10. Effects of MnSO, on K, values of glucose-6-P. Glycogen synthase in extract of confluent L6 cells was assayed. Net enzyme activity (V ~ V,) is activity in the presence of glucose-62 substracting activity in the absence of glucose&P. The concentrations of MnSO, were: 0 mM, 0; 2.5 mM, 0; 5 mM, and 8 mM, n .

glucose-&P, the A values are 2.1, 1.4, and extract. Similar results have been reported 0.5 mM respectively. These A,,, values are for cultured choriocarcinoma cells (11) calculated from net maximal activities where the conversion of glycogen synthase, which are the differences between the maxi- but not of phosphorylase, was missing. mal activity and the activity at zero concen- Similar to choriocarcinoma cells, L6 cells tration of activator. The activator-saturasynthesized glycogen in the presence of Dtion curves for glucose-6-P are shown in form synthase. These properties were very Fig. 10. At low concentrations of glucose-6 different from those observed for cultured P, activation of synthase by MnSO, is very brain cells (9), where both glycogen impressive. In the absence of MnSO,, the K, synthase and phosphorylase activities varied value for glucose-6-P is 2.5 mM. In the with glucose concentration in the medium. D-Form glycogen synthase is usually conpresence of 2.5,5 or 8 mM MnSO,, K, values for glucose-6-P are greatly reduced to 0.57, sidered to be inactive at physiological concentration of glucose-6-P. Since only D0.5, and 0.67 mM respectively. form glycogen synthase is present in L6 cells during glycogen synthesis, it appears DISCUSSION that activated D-form synthase, rather than Cell cultures of myogenic lines are now converted I-form synthase, is actually widely used for studies related to cell responsible for glycogen synthesis. We do differentiation (4, 25). These cells retain not know the exact mechanism of activation their capacity for differentiation even after in L6 cells. The concentration of glucose-6-P very long periods of proliferation (26,27). In in the cells is 0.195 mM at peak accumulation the current study, myoblasts of L6 cell line of glycogen and varies between 0.02 mM and proliferated normally as reflected by ex- almost zero from 18 to 24 h after fluid pected increases in protein and glycogen change (assuming 11% wet weight is contents. After reaching confluency, fusion protein, and 60% of the wet weight is and, consequently, formation of dense water). Since the K, value for glucose-6-P is multinucleated fibers, were observed micro- 2.5 mM (Fig. lo), activation of D-form scopically. However, glycogen synthase synthase by glucose-6-P alone may not be and phosphorylase of cell extracts were sufficient for the observed glycogen syntheentirely in phosphorylated form whether sis. However, D-form synthase in cell extracts were prepared from proliferating extract was also activated by the combinamyoblast cultures or from confluent myotube tion of Mn2+ and SO;-, and the activation cultures. This might be due to complete lack was very prominent at low concentration of of dephosphorylation reactions in the crude glucose-6-P. The K, value for glucose-6-P in

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the presence of 2.5 or 5 mM MnSO, is only 20% of the control value (Fig. 10). Physiological significance of Mn*+ and SO;- in the regulation of glycogen metabolism is not known. D-Form synthase in choriocarcinoma cells is also activated by MnSO, (11). Lack of conversion of glycogen synthase has also been reported in liver extracts of adrenalectomized and fasted rat (28, 29>, alloxan diabetic rat (30), and in mouse Ehrlich ascites carcinoma (31). In choriocarinoma cells and liver extracts of adrenalectomized and fasted rats, the lack of conversion was due to the alteration of synthase D, which was no longer a substrate of phosphatase. We do not know whether synthase D in L6 extract has been altered in any way, but it is readily converted by purified rabbit muscle phosphatase (Fig. 3). In addition, pellet enzymes recovered from high-speed centrifugation are also converted in the presence of MnCl,. Therefore, the lack of conversion of L6 enzymes is probably because of phosphatase inhibitor(s). In mouse ascites carcinoma (31), lack of conversion was also due to a synthase phosphatase inhibitor, which did not however, inhibit phosphorylase conversion. Chromatographic separation of L6 inhibitor revealed two main types of inhibitors. Inhibitor eluted at lower salt concentration is relatipely unaffected by conditions that favor phosphorylation while that eluted at higher salt concentration is activated by phosphorylation. The relative elution positions of these two inhibitors are different from those found in rabbit (14) and rat skeletal muscle, adrenal cortex, and beef heart (32). Further purification will be required for the comparison of inhibitors from such varied sources. Several separate studies suggested that the action of insulin in glycogen synthesis is through synthase activating enzyme or, more specifically phosphatase (29,30,33). Phosphatase activity is very low in heart (34) and liver extracts (30, 35) of diabetic rats, and insulin administration restored the enzyme to normal levels. In streptozotocin-induced diabetic rat liver, phosphatase activity was lowered, and there was an elevation of phosphatase inhibitor activity (15). Insulin treatment resulted in elevation

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of phosphatase and reduction of inhibition (15). However, the exact regulation of phosphatase and its inhibitor activities is not known, and other insulin effects observed in many laboratories with different animal models are variable with respect to time sequence and glucose dependency (30, 33, 36-39). It is not known whether L6 cultures respond to insulin in the activation of glycogen synthesis, but it is known that insulin stimulates fusion and differentiation of L6 myoblasts (8). In addition, L6 cultures resemble laboratory-induced diabetic animals in that they both lack the dephosphorylation reaction. Therefore L6 cell cultures studied in this paper may provide an ideal isolated system for studying the involvement of phosphatase inhibitor in the hormonal regulation of glycogen metabolism. Using this culture system, measurement of direct effects of hormone and/or metabolites is possible, and secondary responses to hormone, as encountered in intact animals, are avoided. ACKNOWLEDGMENT We thank Dr. Janice Y. Chou (LBS, NIH) for her advice in growing and maintaining L6 cells in culture. REFERENCES 1. YAFFE, D. (1968) Proc. Nat. Acad. Sci. IJSA 61,477-483. 2. YAFFE, D. (1969) Curr. Top. Develop. Biol. 4, 33-77. 3. LOOMIS, W. F., WAHRMANN, J. P., AND LUZZATI, D. (1973) Proc. Nat. Acad. Sci. USA 70, 425-429. 4. YAFFEE, D., and SAXEL, 0. (1977) Differentiation 7, 159-166. 5. WAHRMANN, J. P., LUZZATI, D., AND WINAND, R. (1973) Biochem. Biophys. Res. Commm. 52, 576-581. 6. WAHRMANN, J. P., WINAND, R., AND LuzZATI, D. (1973) Nature New Biol. 245, 112-113. 7. TARIKAS, N., AND SCHUBERT, D. (1974) PTOC. Nat. Acad. Sci. USA 71, 2377-2381. 8. MANDEL, J. L., AND PEARSON, M. L. (1974) Nature New Biol. 251, 618-620. 9. PASSONNEAU, J. V., AND CRITES, S. K. (1976) J. Biol. Chem. 251.2015-2022. 10. GLINSMANN, W., PAUK, G., AND HERN, E. (1970) Btichem. Biophys. Res. Commun. 39, 774-782. 11. HUANG, K.-P., CHEN, C. H.-J., AND ROBIN-

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