Regulation of Glycogen Metabolism in Astrocytoma and Neuroblastoma Cells in Culture

The regulation of glycogen metabolism in C-6 astrocytoma and C-1300 neuroblastoma cells in culture has been investigated. of control

The regulation of glycogen metabolism in C-6 astrocytoma and C-1300 neuroblastoma cells in culture has been investigated.
Two modes of control of glycogen metabolism appear to be operative. The regulation of intracellular glycogen concentrations and the predominant forms of glycogen phosphorylase and glycogen synthase vary with (a) the available energy supply, and (b) altered intracellular concentration of cyclic adenosine 3':5'-monophosphate (cyclic AMP). Both cell lines respond to glucose in the medium; when glucose levels are high, glycogen is synthesized, glycogen phosphorylase a decreases, and glycogen synthase a increases. When glucose in the medium decreases to a critical level, the phosphorylase a increases and glycogen concentrations in the cells decrease in parallel with the medium glucose. The critical glucose concentration is 2.5 rnb: for the astrocytoma cells and 4 mM for the neuroblastoma cells. Insulin promotes the conversion of phosphorylase to the b form and synthase to the a form in both cell lines. All of these changes occur without alteration in the intracellular cyclic AMP concentrations.
When cyclic AMP concentrations are increased in either cell line, phosphorylase a is increased, synthase a is decreased, and glycogen concentrations decrease. Isobutyl methylxanthine is effective in promoting glycogenolysis in both cell lines. Norepinephrine is effective with the astrocytoma cells, and prostaglandin E, is effective with the neuroblastoma cells.
Several investigations have been made of glycogen metabolism in brain, with regard to the effect of various experimental procedures on glycogen turnover, accumulation, and degradation (l-6). One of the considerable problems in evaluating results observed with whole brain is that both neuronal and glial elements are involved. This is particularly important when one is considering glycogen metabolism, since many investigators consider that the glycogen is largely confined to glial elements, and particularly astrocytes (7). However, it has been shown in analyses of single cells that glycogen is present in at least some of the large neurons of the central nervous system (8). The availability of astrocytoma and neuroblastoma cells in culture affords an opportunity to study separately the glycogen metabolism in cells of neuronal and glial origin. The synthesis and breakdown of glycogen in cultures of C-6 astrocytoma cells and C-1300 neuroblastoma cells have been examined under normal growth conditions and in the presence of glucose 10 times greater than that in the usual medium. A cycle of reciprocal changes in the active forms of glycogen phosphorylase and glycogen synthase occurred when fresh medium was added to cells in the stationary phase of culture. These changes were accompanied by changes in the concentrations of intracellular glucose, glucose-6-P, UDP-glucose, and glycogen, but not of cyclic adenosine 3':5'-monophosphate. Furthermore, the administration of insulin increased the active form of glycogen synthase and decreased the amount of phosphorylase in the active form, without changes in cyclic AMP.' The changes effected by insulin are more pronounced when glucose is added at the same time in the C-6 astrocytoma cells.
The effects of agents which did cause increases in cyclic AMP concentrations in the cells were also exami:led. Norepinephrine was effective in increasing the intracellular cyclic AMP concentrations in the astrocytoma cells. Isobutyl methylxanthine had a somewhat marginal effect on cyclic AMP, but did increase the active form of glycogen phosphorylase and diminish synthase a.' In the neuroblastoma cells prostaglandin E, and isobutyl methylxanthine caused an increase in intracellular cyclic AMP. In both cell lines, when cyclic AMP concentrations were elevated, phosphorylase was activated, synthase a was decreased, and glycogen content decreased. MATERIALS   and freezing in liquid nitrogen. The cells were then suspended in 1 ml of ice-cold 0.03 N HCl. An aliquot was removed for glycogen analysis, to which was added sufficient alkali to make the preparation 0.1 N NaOH, and heated for 10 min at 100". This procedure destroys the glucose present which would otherwise be measured with the end product in the glycogen assay (17). The remainder of the suspension was centrifuged and the supernatant extract used for the analysis of the other compounds.
The analysis for UDP-glucose was conducted within a few hours of sample preparation, and the samples were kept at 0". UDP-glucose is not stable in 0.03 N HC1 if the sample is heated or frozen. Recovery is complete, however, after 5 hours at 0".  Fig. 1). Within 5 min after addition of fresh medium, the intracellular glucose concentration was 80-fold greater than that 24 hours after feeding. Glucose-6-P also rapidly increased in the cells and to an even greater degree. UDP-glucose increased more slowly to a peak Concentrations of metabolites and cyclic AMP in C-6 astrorytoma cells at intervals after feeding The cells were grown as described under "Materials and Methods." On the day of the experiment, the medium was removed, fresh medium with serum added, and the cells were frozen at the stated intervals as described under "Materials and Methods." The number of dishes analyzed at each interval is given in parentheses and the means * S.E. are given when more than 2 dishes were used. The enzymes and metabolites were measured as described under "Materials and Methods." as a function of time after feeding. C-6 cells were grown for 10 days as described under "Materials and Methods." The old medium was removed, and fresh medium containing serum was added. Medium samples were taken and the cells frozen at the times indicated.
level 5 to 7 hours after new medium was added. The concentration of glycogen was increased at 15 min after feeding and continued to increase until 3 hours after feeding. When glucose in the medium reaches about 2.5 mM the glycogen begins to decrease. This observation was repeated several times, and a glucose concentration in the medium of 2 to 3 mM appears to be critical; at this point glucose decreases, and glycogen decreases in a parallel fashion (Fig. 1). The amount of glycogen synthase in the a form was 25% or greater until 30 min after feeding, when the active form began to decrease to a minimum of 4% at Glycogen Metabolism in Astrocytoma and Neuroblastoma Cells The cells were grown and treated as in Table I. The enzyme activities were analyzed as described in the text. The number of dishes at each interval is given in parentheses, and the values expressed as means * S.E. when more than 2 samples were analyzed.
The synthase a and b activities show striking differences when plotted against intracellular glycogen concentration (Fig. 2). As glycogen increased to the peak level at 3 hours ( Fig. 1). the amount of synthase in the a form as well as the percentage decreased, while the amount of b varied only 25%. The decrease in activity of the a form can, in part, be explained by the inhibition of synthase phosphatase activity by glycogen (18,19). However, the decrease in total amount of synthase activity (Table  II) is apparently due to the formation of an inactive form of synthase (see under "Discussion"). Because the concentration of extracellular glucose appeared to be critical, the effect of increased glucose in the medium was investigated.
When medium glucose was 50 mM, higher concentrations of intracellular glucose were seen than with 5 mM glucose (Table  III, cf. Table  I  The cells were grown as described under "Materials and Methods." On the 10th day of culture, fresh medium without serum was added and 10 min later 0.1 rn~ norepinephrine was added. The cells were incubated for 20 or 35 additional min, the medium removed, and the ceils frozen and prepared for analysis. The number of dishes in each group is shown in parentheses and values are given as the means i S.E. The cells were grown as described under "Materials and Methods," and the treatment was as described for C-6 cells in Table I. Values are the mean of 2 to :I dishes (in parentheses) at each time interval L S.E. when appropriate.
The cells were prepared and analyzed as described in Table I  to those of the astrocytoma cells. Glycogen increased after feeding for 2 hours, but the highest level was less than one-half that found in C-6 cells. The amount of phosphorylase in the a form decreased after feeding and increased again as glucose in the medium decreased (Table  VIII), Glycogen synthase in the active form increased slightly and fell to a minimum when the glycogen concentration in the cell was highest.
As with phosphorylase, as medium glucose decreased, the synthase a again increased. Total synthase activity varied almost P-fold, a slightly smaller range than seen in C-6 astrocytoma cells (Table  II)  The cells were grown as described under "Materials and Methods." units of insulin. After 20 min the medium was removed and the cells On the 10th day of culture, fresh medium without serum (7 ml) was frozen and extracted or homogenized as described under "Materials added and cells incubated for 3 or 5 hours. Two milliliters of phosphate-and Methods." The number of dishes is given in parentheses, and the buffered saline or Dulbecco's modified Eagle's medium was added + 5 values expressed as means + S.E. when appropriate. The increase in cyclic AMP concentration was more than additive and the changes in phosphorylase a and synthase a were also accentuated.
In addition, there was a substantial decrease in glycogen as well as in glucose and glucose-6-P. DISCUSSION The

glucose.
Initially, the amount of synthase in the active form was high; however, glycogen continued to increase even as the active enzyme was diminished (Tables  I and II, Fig. 1). There appeared to be a maximum level of glycogen which is in some way regulated in the cell. The regulation may be due in part to the glycogen concentration which regulates the amount of synthase a (Fig. 2), presumably by inhibiting synthase phosphatase (18,19). In the astrocytoma cells, glycogen reached essentially the same level whether in the presence of 5 or 50 mM glucose (Fig. 1, Table  III). The maximum concentration of glycogen in the neuroblastoma cell line was about one-half that in the astrocytoma cells. Synthase activity in the neuroblastoma cells was one-third to one-half that in the astrocytoma cells. The amount of enzyme in the cell may in part regulate the glycogen levels, since in both cell lines the rate of glycogen accumulation was close to the synthase a activity (see below). In both cell lines, when a certain glucose concentration of the medium was reached, glycogen content in the cells began to decrease.
As the medium glucose fell, phosphorylase was activated and the glycogen content of the cells decreased. The amount of synthase a also increased at a time when glycogen stores were being depleted (Tables  I and II, Fig. 1). It appears that when available energy supplies are low, the regulation of and Neuroblastoma Cells 2021 values for transport in both cell lines are well below the medium glucose concentration, 1.7 mM glucose for C-6 cells, and 1 mM glucose for neuroblastoma cells (28). The maximal rate of glycogen synthesis in the astrocytoma cells occurred between 5 and 15 min after feeding and was equivalent to 186 nmol/mg of protein/hour ( Fig. 1). This is remarkably close to the synthase a activity 15 min after feeding, which is 172 nmol/mg of protein/hour (Table II). In the neuroblastoma cell line, the maximal rate of synthesis was 48 nmol/mg of protein/hour between 20 and 30 min after feeding (Table VII). The measured synthase a activity at 20 min after feeding was 41 nmol/mg of protein/hour. At other time periods, the correlation between the rate of glycogen accumulation and synthase a activity is similar. The conditions in the cell must be such that synthase a can operate at maximum velocity in both cell lines at these times. It is of interest to note that with normal medium, total synthase activity in the C-6 cells decreases up to 5 hours after feeding, and this is due almost entirely to a decrease in synthase a (Table II, Fig. 2). After feeding with 50 mM glucose, there is a striking decrease in both synthase a and total synthase. In the C-1300 neuroblastoma cell line, there is also a decrease in synthase activity, although less pronounced than in the C-6 cells ( Table VIII). The cause of the loss of total enzyme activity is not known. The enzyme could be degraded, but this seems unlikely to occur so rapidly, as the synthase of astrocytoma cells after 3 hours in 50 mM glucose is 28% of the initial amount (Table III). Furthermore, when neuroblastoma cells are refed, the synthase activity is increased 1.4-fold in 10 min (Table VIII). The cells appear to be viable and the phosphorylase activity remains constant. The loss of activity of synthase may be related to the phenomenon of inactive forms of glycogen synthase observed by others. There appears to be at least two main types of inactive enzyme.  (32,33). This enzyme species is postulated to be more phosphorylated than the a form but less than the b form. In addition, the b form has been made inactive by incubation with ATP and Mg*+ and is thought to be "extraphosphorylated" (33). It is attractive to consider that when the cells are in a medium with plenty of available nutrient and glycogen concentrations are high, a mechanism may exist that not only regulates the amount of enzyme in the active form, but can reduce the amount of total enzyme activity available without protein degradation.
The inactive species may have extra phosphate groups, formed while energy stores are high. A further possibility is that a glycogen.enzyme inactive complex is formed. In the C-6 cells at least, the synthase activity is lowest when glycogen stores are highest, and the cells presumably are in a "saturated" state. The synthase.glycogen complex may become inactive when glycogen levels are very high, if all the enzyme is tightly bound.
The effects of insulin are similar in both cell lines, although the changes observed in metabolite levels in C-6 cells were not seen in the neuroblastoma cells. Furthermore, the changes were more marked in general when glucose was added with the insulin (Tables   IV and IX). All of these effects occurred without alteration in the cyclic AMP concentration.
In studies with perfused livers, Miller and Larner (23) associated insulin action with decreased cyclic AMP concentrations and decreased protein kinase activity. However, the decreased cyclic AMP was observed only if the livers were treated consecutively with glucagon and insulin, and not when insulin alone was used. It is possible that some subtle changes occur in a small pool of cyclic AMP in the cells (34); it seems more likely that the insulin effect is related to the metabolic effects demonstrated with glucose. Increases in synthase a activity following insulin administration have been observed by others in liver (35)(36)(37)(38), diaphragm (39-42), skeletal (43) and heart muscle (44,45), and adipose tissue (46).
In the C-6 cells, the mode of effect of isobutyl methylxanthine with or without adenosine on the phosphorylase and Glycogen Metabolism in Astrocytoma and Netiroblastoma Cells synthase activities and glycogen concentration is not clear. In one experiment cyclic AMP was not changed, and in a second, the concentration of cyclic AMP were about doubled (Table  VI). In other experiments done at different time intervals after feeding, cyclic AMP was consistently elevated by this treatment (26). It seems likely that the enzyme changes do reflect an effect on the cyclic nucleotide.
No changes were ever observed with adenosine alone.
The neuroblastoma cell line responded to prostaglandin E, and isobutyl methylxanthine with increased cyclic AMP concentrations, and concomitant changes in metabolites and the forms of phosphorylase and synthase (see under "Results" and Table X). All of the changes were more marked when both compounds were given, as would be expected since prostaglandin E, stimulates adenylate cyclase, and isobutyl methylxanthine inhibits phosphodiesterase.
In conclusion, there appears to be at least two ways of regulating glycogen metabolism in these cell lines. The active forms of synthase and phosphorylase vary with the glucose content of the medium and as a consequence, either form or degrade glycogen. When glucose is added to starved cells, glycogen synthesis is turned on; as glucose disappears from the medium, glycogenolysis is favored. All of these events occur without discernible changes in cyclic AMP concentrations. However, if agents are used which increase the cyclic AMP in the cells, phosphorylase a is increased, synthase a is decreased, and glycogenolysis is favored.