1 Metabolic Research Laboratory and Section of Endocrinology, Metabolism and Nutrition, Minneapolis Veterans Affairs Medical Center, and Departments of 2 Medicine and 3 Food Science and Nutrition, University of Minnesota, Minneapolis, Minnesota 55417
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ABSTRACT |
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We have reported that glycogen synthesis and degradation can occur in vivo without a significant change in the amount of phosphorylase a present. These data suggest the presence of a regulatable mechanism for inhibiting phosphorylase a activity in vivo. Several effectors have been described. AMP stimulates, whereas ADP, ATP, and glucose inhibit activity. Of these effectors, only the glucose concentration changes under normal conditions; thus it could regulate phosphorylase a activity in vivo. We previously have reported that, when all of these effectors were present at physiological concentrations, the net effect was no change in phosphorylase a activity. Addition of caffeine, an independent inhibitor of activity, to the above effectors not only resulted in inhibition but also restored a glucose concentration-dependent inhibition. Because uric acid is an endogenous xanthine derivative, we decided to determine whether it had an effect on phosphorylase a activity. Independently, uric acid did not affect activity; however, when added at a presumed physiological concentration in combination with AMP, ADP, ATP, and glucose, it inhibited activity. A modest but not statistically significant glucose concentration-dependent inhibition was also present. Thus uric acid may play an important role in regulating phosphorylase a activity in vivo.
glucose; glycogen metabolism; purine metabolism; xanthine; multiplex enzyme regulation
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INTRODUCTION |
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IN LIVER, the glycogen phosphorylase a activity assayed independently of known effectors is ~10- to 20-fold greater than the glycogen synthase R activity (active form) (7, 24). The phosphorylase a is also sufficient to degrade most of the glycogen present within a matter of minutes. Thus it is apparent that inhibition of phosphorylase a activity in vivo must be present. In addition, glycogen synthesis and degradation occur normally without a change in the proportion of total phosphorylase in the a-form (7). The latter implies the presence of regulatable effectors of phosphorylase a activity.
We (8) and others (16, 21, 22) have previously demonstrated that AMP stimulated, whereas ADP, ATP, and glucose inhibited phosphorylase a activity. UDP-glucose, glucose 6-phosphate, and fructose 1-phosphate also were weak inhibitors at physiological concentrations. We have reported that, in the presence of a combination of all of these effectors at concentrations likely to be present in vivo, the stimulatory and inhibitory effects canceled each other (8). The net effect was that activity was unchanged or was modestly inhibited compared with the phosphorylase a activity assayed without addition of any effector. AMP, ADP, and ATP are quite stable in vivo (17, 18), except under pathological conditions; however, the physiological concentration of glucose varies considerably (19). Thus glucose may be a potential regulator of activity in vivo. The intracellular glucose concentration varies between 8 and 20 mM (19). However, in the presence of the other known effectors, glucose in this concentration range did not change the activity. Therefore, under these conditions, glucose was not regulating.
In a previous study, addition of caffeine (1,3,7-trimethylxanthine) both inhibited activity and partially restored the sensitivity of the enzyme to inhibition by glucose at concentrations within the normal range (10). Caffeine also allowed the inhibitory activity of ADP, and to a lesser degree that of ATP, to be expressed. The observation that caffeine inhibited activity led to the speculation that an endogenous effector that binds to the phosphorylase enzyme at the same site as caffeine is present in vivo (14); nevertheless, none had been identified. Therefore, we decided to determine whether uric acid, an endogenous xanthine analog of caffeine, is the natural ligand that inhibits phosphorylase a activity in a regulatable fashion and/or allows inhibition by glucose to occur.
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EXPERIMENTAL PROCEDURES |
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[32P] Pi was purchased from Amersham-Pharmacia-Biotech (Piscataway, NJ). All chemicals were obtained from Sigma Chemical (St. Louis, MO). The rabbit liver glycogen used was purified by passage through a mixed-bed ion exchange resin (Amberlite MB-3; Mallinckrodt Laboratory Chemicals, Phillipsburg, NJ).
Male Sprague-Dawley rats, weighing 130-220 g, were purchased from Bio-Lab (Madison, WI) and were the source of liver for phosphorylase purification.
Glycogen phosphorylase a was purified essentially to homogeneity, as described by Tan and Nuttall (23), with only minor modifications as described previously (8). The specific activity was 22 units/mg protein under the conditions of the assay. A unit represents 1 µmol of product produced/min.
During purification, liver phosphorylase a and total phosphorylase activities were monitored in the direction of glycogen synthesis by the Tan and Nuttall (23) modification of the method of Gilboe et al. (11). In all subsequent studies, the phosphorylase a activity was measured in the direction of glycogenolysis as described previously (8, 10). In this method, phosphorylase a is determined by incubating purified phosphorylase in the presence of glycogen and [32P]Pi. The radioactivity incorporated into glucose 1-phosphate is then measured.
Phosphorylase activity was stable at 37°C; the velocity of the reaction was linear with time and the amount of phosphorylase added. Over the 3-min incubation time period used in the assay, only 0.05% of the substrate was converted into product. Thus the conditions approximated an initial velocity (data not shown).
Pi concentrations of 1 and 5 mM were used, because they are
likely to represent the range of free Pi concentrations in
the liver (2). The Michaelis-Menten constant for
Pi is 1.1 mM at a saturating glycogen concentration as
used in the present assay (8). The phosphorylase
a activity in the absence of effectors was 28 ± 1.6 µmol glucose 1-phosphate
produced · min1 · ml
1 at a
5 mM Pi concentration and 11 ± 1.5 µmol · min
1 · ml
1 at a 1 mM Pi concentration (control activities).
Uric acid was solubilized using LiCO3 (the stock solution contained 5 mM uric acid and 0.5 mM LiCO3). The final concentration of LiCO3 in the assay mixture varied between 0.05 and 0.1 mM. Concentrations of LiCO3 as high as 1 mM in the final assay mixture did not affect phosphorylase activity (data not shown).
Data are presented as means ± SE. Statistics were done with two-way ANOVA. When the number of observations taken within each treatment was different, analysis of variance was done for an "unbalanced" design. Comparison of treatment means with the control was done by Dunnett's procedure. P < 0.05 was the criterion for statistical significance.
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RESULTS |
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Combination 1 is a mixture of 0.3 mM AMP, 3 mM ADP, 6 mM ATP, 0.5 mM UDP-glucose, 0.3 mM glucose 6-phosphate, 0.3 mM fructose 1-phosphate, and 8 mM glucose. These are the physiological concentrations of the effectors present in liver intracellular water (18). Combination 2 represents the same combination of effectors, but the glucose concentration was increased to 20 mM. Thus the two combinations encompass the glucose concentrations estimated to be present in vivo (19).
When uric acid at concentrations varying from 0.025 to 3 mM was added
to combination 1 or 2, inhibition of the activity
was observed. The inhibition was dependent on both uric acid and
glucose concentration. With combination 1, the activity at
reported physiological concentrations of uric acid (0.05-0.3 mM)
(1, 6) was reduced to 70 (uric acid concentration 0.05 mM)
and 48% (uric acid concentration 0.3 mM) of control activity at a 1 mM
Pi concentration. At 5 mM Pi, it was reduced to
79 and 57% of the control, respectively (Fig.
1A). With combination
2, the activity was reduced to 65 and 41% at a 1 mM
Pi concentration and 72 and 51% of the control activity at
a 5 mM concentration, respectively (Fig. 1B).
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The I0.5 for uric acid inhibition under these conditions was ~0.3 mM in combination 1 and ~0.2 mM in combination 2. The inhibition was largely independent of the Pi concentration, i.e., uric acid appeared to affect primarily the maximal velocity. There was little inhibition when uric acid was added alone (Fig. 1C).
When uric acid at a 0.2 mM concentration was added to the combination of effectors and the glucose concentration was varied between 8 and 20 mM, increasing the concentration of glucose further inhibited activity but only modestly (Fig. 1D).
The ADP concentration used in the preceding experiments (3 mM) was that
determined chemically in a liver tissue extract. NMR data, however,
suggest that the cytosolic concentration may actually be significantly
lower (3-5, 13, 15). The ADP concentration in the
liver cannot be determined directly by NMR; rather, it is determined
indirectly by calculating the difference in areas between the -peak
of ATP (which contains a contribution from the
-phosphate of ADP)
and the
-peak of ATP (which does not contain an ADP component). For
low ADP levels, this measurement is not very accurate, because the NMR
measurement of ADP relies on determination of a small difference
between two large ATP peaks. Furthermore, NMR is not very sensitive in
detecting molecules present at <0.3 mM in vivo (15); in
addition, other nucleoside diphosphates may contribute to the peaks.
Values in the range of 0.05-0.2 mM have been reported for free ADP
concentration in the liver (3-5, 13, 15). This free
ADP concentration may be the fraction that influences the phosphorylase
a activity; therefore, we repeated the experiments using the
same combination of effectors but with the ADP concentration reduced to
0.1 mM. In these experiments, combinations 1 and
2, in the absence of uric acid, modestly inhibited phosphorylase
a activity. The activity with combination 1 was
83% of control activity at 1 mM Pi concentration and 89%
of control activity at 5 mM Pi concentration. With
combination 2, the activity was 76 and 79% of control at 1 and 5 mM Pi concentrations, respectively.
When uric acid at concentrations varying from 0.025 to 2 mM were added
to combinations 1 or 2 where the ADP
concentration was 0.1 mM, inhibition of phosphorylase a
activity again was observed. With the use of combination 1,
the activity at a uric acid concentration of 0.3 mM was reduced to 58%
of control at 1 mM Pi concentration and to 61% of the
control at 5 mM Pi concentration (Fig.
2A). With combination
2, the activity was reduced to 41 and 52% of control at 1 and 5 mM Pi concentrations, respectively (Fig. 2B). The inhibition was similar to that found when the ADP concentration in
the combination of effectors was 3 mM. With the lower ADP
concentration, the I0.5 for uric acid inhibition was ~0.8
mM for combination 1 at both 1 and 5 mM Pi
concentrations and ~0.7 and 1.2 mM for combination 2 at 1 and 5 mM Pi concentrations, respectively. When 0.3 mM uric
acid was added to the combination of effectors with 0.1 mM ADP and the
glucose concentration was varied between 8 and 20 mM, the increasing
concentration of glucose again further inhibited phosphorylase
a activity modestly (Fig. 3).
The decrease did not quite reach statistical significance
(P = 0.06).
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Because uric acid alone was not inhibitory but did inhibit in the presence of a combination of effectors, we were interested in determining the interaction of uric acid with each specific effector.
Glucose is an independent inhibitor of phosphorylase a. When
glucose was present at an 8 mM concentration, addition of uric acid at
concentrations of 0.1-0.3 mM also had no effect on the glucose
inhibition (Table 1; P > 0.05). In the presence of a 20 mM glucose concentration, addition of
uric acid actually reduced the glucose inhibition of phosphorylase
a activity (Table 1; P < 0.05 vs. that in
the absence of uric acid).
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ADP is an independent inhibitor of phosphorylase a activity (8). When uric acid at concentrations from 0.1 to 0.3 mM was added to 3 mM ADP, the activity was reduced to ~59 and 78% at 1 mM and 5 mM Pi concentrations, respectively. This was as expected from the addition of 3.0 mM ADP alone, i.e., an interaction between uric acid and this effector was not present (Table 1; P > 0.05 vs. that in the absence of uric acid).
ATP is also an independent inhibitor of phosphorylase a activity, although it is a weaker inhibitor than ADP (8). When uric acid (0.1-0.3 mM) was added to ATP (6 mM), the inhibition of phosphorylase a activity was similar to that of 6 mM ATP alone (P > 0.05). That is, uric acid again did not have an additional inhibitory effect (Table 1).
AMP stimulates phosphorylase a activity and antagonizes glucose inhibition (8). The presence of uric acid did not affect the stimulation of activity by AMP (Table 1; P > 0.05). When uric acid was added to AMP and glucose (8 or 20 mM) together, the stimulation of phosphorylase activity in the presence of uric acid was similar to that observed in its absence, i.e., addition of uric acid to AMP with or without glucose had no further effect (Table 1; P > 0.05). The addition of glucose reduced AMP stimulation at a 1 mM Pi concentration, as expected (8).
When uric acid (0.1-0.3 mM) was added to 3 mM ADP with 8 mM glucose, or when it was added to 6 mM ATP with 8 mM glucose (Table 1), an independent inhibitory effect of uric acid again was not seen (P > 0.05). This was also true when uric acid was added to a combination of 6 mM ATP and 20 mM glucose (Table 1; P > 0.05). However, when uric acid was added to 3 mM ADP and 20 mM glucose, a biphasic effect was seen (Table 1; P < 0.05). At a low concentration of uric acid (0.1 mM), the inhibitory effect of ADP and glucose was diminished; at a higher concentration (0.3 mM), it was not diminished.
When uric acid was added to the combination of AMP and ADP, an
additional inhibition by uric acid was not present (Fig.
4A; P > 0.05). However, when uric acid was added to the combination of AMP (0.3 mM), ADP (3 mM), and glucose (8 or 20 mM), the uric acid inhibitory
effect was expressed (Fig. 4, B and C;
P < 0.05 for values at 0.3 mM uric acid concentration
vs. those in the absence of uric acid), i.e., the presence of ADP, AMP,
and glucose were necessary for the expression of the uric acid effect;
ATP was not necessary. Interestingly, when ATP was added with AMP, an
inhibition by uric acid was also observed (Fig. 4D;
P < 0.05).
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In summary, when uric acid was added to AMP, ADP, or ATP alone, the effect observed was similar to that in the absence of uric acid, i.e., the concentration-dependent inhibitory effect of uric acid was not observed. At high concentrations of glucose, inhibition by glucose was actually diminished by low conditions of uric acid. The inhibitory effect of uric acid was observed only in the presence of AMP, ADP, and glucose (Fig. 4, B and C) or in the presence of AMP, together with ATP (Fig. 4D). Glucose 6-phosphate (0.3 mM), fructose 1-phosphate (0.3 mM), and UDP-glucose (0.5 mM) were not required for the inhibition by uric acid to be expressed, i.e., the inhibitory effect of uric acid in the presence of AMP, ADP, and glucose, or in the presence of AMP and ATP, was similar whether glucose 6-phosphate, fructose 1-phosphate, or UDP-glucose was present.
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DISCUSSION |
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The present data demonstrate that uric acid did not affect phosphorylase a activity when added alone at concentrations reported to be present in the liver (1, 6). However, when added in the presence of a combination of effectors, it inhibited activity rather strongly and in a concentration-dependent manner. The inhibition was similar when the ADP concentration in the combination of effectors was high (3 mM) or low (0.1 mM). In addition, changes in glucose concentration within the physiological range further reduced the activity; however, the decrease did not quite reach statistical significance. These data suggest that the ligand that binds to the purine nucleoside site (caffeine site) may be uric acid acting in concert with other effectors. Thus uric acid, as well as glucose, may be a potential regulator of liver phosphorylase a activity in rats in vivo. Hypoxanthine and xanthine, which are endogenous products of purine metabolism, may also contribute to the inhibition in vivo (10), but this remains to be determined.
For uric acid inhibition to be expressed, AMP, ADP, and glucose were required (Fig. 4, B and C). ATP in association with AMP may also allow uric acid inhibition to be expressed (Fig. 4D). That is, there is a synergistic interaction between the xanthine derivatives and AMP, ADP, and glucose, and possibly ATP, a very complex regulatory mechanism. This we refer to as a multiplex site-site regulation of enzymic activity.
The present data may potentially link purine metabolism to glycogen metabolism and help explain why phosphorylase a, present in amounts in vivo that should rapidly degrade glycogen, does not do so.
The uric acid concentration in the rat liver was reported to be 0.3 mM in one study (1) and 0.1 mM in another (6). Whether the uric acid concentration changes under physiological conditions or with exogenous administration of hormones is largely unknown. In this regard, it has been reported (20) that urate formation is rapidly stimulated by sympathetic nerve stimulation or by the addition of glucagon or norepinephrine to a perfused rat liver preparation. The concentration of urate in the liver was not determined; thus, whether a significant change in hepatic uric acid concentration or the concentration of other xanthines occurs with hormonal stimulation remains to be determined. If so, this would be a unique regulatory mechanism for controlling glycogen degradation. Others (12) have reported that glucagon or cAMP increases the level of 5-phosphoribosyl-1-pyrophosphate in hepatocytes. The concentration of this purine nucleotide precursor is considered to be important in determining the rate of purine synthesis. If uric acid is an important inhibitor of phosphorylase a in vivo, a rise in the concentration of these compounds as a result of increased synthesis should oppose the glycogenolytic activity of an increased phosphorylase a stimulated by glucagon. This represents a paradoxical response. However, the increase in uric acid could be transient, as observed for the release of uric acid with nerve stimulation in the perfused liver preparation (20). This could then explain our observation that glycogenolysis is delayed despite an immediate increase in phosphorylase a activity when glucagon is administered to rats (9). These purine derivatives may also interact with intrahepatic glucose in regulating this process. This remains to be explored.
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ACKNOWLEDGEMENTS |
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We thank Claudia Durand for excellent secretarial assistance.
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FOOTNOTES |
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This study was supported by an Advanced Research Career Development Award and Merit Review Research Funds from the General Medical Research Service, Department of Veterans Affairs, and by Grant RO1-DK-43018 from the National Institute of Diabetes and Digestive and Kidney Diseases.
Address for reprint requests and other correspondence: N. G. Ercan-Fang, Minneapolis VA Medical Center, One Veterans Drive (111G), Minneapolis MN 55417 (E-mail: ercan001{at}tc.umn.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 June 2000; accepted in final form 5 October 2000.
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