Introduction

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Phosphorylase is present in liver in a phosphorylated and unphosphorylated form referred to as phosphorylase a and b, respectively. Only the a form is active at substrate concentrations present in vivo (Tan and Nuttall, 1975). Traditionally, glycogen degradation has been considered to be regulated by interconversion between the active (phosphorylase a) and inactive (phosphorylase b) forms of the enzyme. However, as pointed out previously, the ratio of phosphorylase a to b does not change significantly with glucose administration either during glycogen synthesis or subsequent degradation (Tan and Nuttall, 1975). Also, the measured phosphorylase a activity cannot explain the changes in glycogen concentration during the normal feeding/fasting cycle (Chen et al., 1992). Thus, regulation of phosphorylase a activity by allosteric effectors is likely to be important in regulating glycogenolysis.

It is known that AMP stimulates liver phosphorylase a activity, whereas ATP, ADP, glucose and fructose-1-P, glucose-6-P and UDP-glucose inhibit its activity. However, when a mixture of these effectors was added to a rat liver phosphorylase a preparation at concentrations reported to be present in liver, the net effect was an activity that was the same as in the absence of any effector (Ercan et al., 1996) .

It has been reported that caffeine strongly inhibits phosphorylase a activity in both liver and skeletal muscle. The inhibition was synergistic with glucose and was relieved by AMP (Kasvinsky et al., 1982, 1978). Therefore, we decided to ascertain the effect of caffeine and other caffeine analogs on liver phosphorylase a activity. We also were interested in how caffeine and its analogs would interact with the other potential effectors identified by us (Ercan et al., 1996), and others (Kihlman and Overgaard-Hansen, 1955, Maddaiah and Madsen, 1966). We were particularly interested in the interaction of caffeine with glucose in the presence of the other effectors. Of the identified modifiers of phosphorylase a activity, only glucose is likely to change significantly under normal conditions. Thus, it potentially could regulate phosphorylase a activity in vivo. A physiological ligand that binds to the caffeine binding site and could synergize with glucose has not as yet been reported. However, some of the structural requirements for a xanthine derivative to act synergistically with glucose have been determined in our study.

	Materials and Methods

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[¹⁴C] glucose-1-P was purchased from New England Nuclear Corporation (Boston, MA); [³²P] inorganic phosphate was purchased from Amersham Corporation (Arlington Heights, IL); sodium secobarbital (Seconal) and glucagon were obtained from Eli Lily (Indianapolis, IN); Q-Sepharose and other chemicals required for phosphorylase purification and assay were purchased from Sigma Chemical Co. (St. Louis, MO) and rabbit liver glycogen was purified by passage through a mixed bed ion exchange resin (Amberlite MB-3) (Mallinkrodt, Inc., Paris, KY).

Male, Sprague-Dawley rats, weighing 130 to 220 g, purchased from Bio-Lab (Madison, WI) were the source of liver for phosphorylase purification. These studies were performed in adherence with the guidelines established in the Guide for the Care and Use of Laboratory Animals (NRC , 1985). Animals were housed in facilities accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC), and the research protocol was approved by the Animal Study Subcommittee of the Minneapolis VA Medical Center, and the University of Minnesota.

Glycogen phosphorylase a was purified essentially to homogeneity as described by Tan and Nuttall (1975) with only minor modifications as described previously (Ercan et al., 1996). The specific activity was 22 U/mg protein under the conditions of the assay. One U 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 (Tan et al., 1975) modification of the method of Gilboe et al. (1972). In all subsequent studies the phosphorylase a activity was measured in the direction of glycogenolysis as described previously (Ercan et al., 1996) .

Phosphorylase activity was stable in the assay mixture at 37^°, pH 7.0. The velocity of the reaction was linear with time and amount of phosphorylase added. Over the 3-min 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).

	Results

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Phosphorylase a activity was determined at a 1- and 5-mM concentration of Pi. These are concentrations that are likely to represent low and high concentrations of free Pi in vivo (Niewoehner and Nuttall, 1988). The chemically measured inorganic phosphate concentration is ~ 5 µM/g wet weight liver (Niewoehner et al., 1984a). However, NMR data suggest that not all of the Pi present is free and thus potentially available to influence enzyme activity. A 1.0 mM free Pi concentration is likely to be a low free concentration and therefore was used in the present experiments. Also, the K_m value for Pi is ~1.1 mM (Ercan et al., 1996). A concentration of 5 mM would be present if essentially all of the Pi measured chemically was free. A 5-mM concentration also is approaching saturation. The control activity in the absence of added effectors was 9.1 ± 0.8 U/ml at 1 mM Pi and 27 ± 1.5 U/ml at 5 mM Pi. A unit of activity is 1 µmol glucose-1-P produced/min under the conditions of the assay.

Caffeine at a 1 mM concentration reduced phosphorylase a activity to only 13% of controls when the Pi concentration was 1 mM. It was reduced to 37% when the Pi concentration was 5 mM (fig. 1A, stippled and solid bars).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effect on phosphorylase a activity of caffeine added with physiological concentrations of glucose, AMP, and ADP. 100% represents the activity in the absence of any effectors. The bars represent the mean ± the SEM (n=5 or greater). The stippled (light) bars represent the activity at a 1 mM Pi (substrate) concentration and the solid (dark) bars the activity at a 5 mM Pi concentration in the presence of caffeine. The clear, bars with a broken line represent the activity in the absence of caffeine.

Glucose at an 8 mM concentration in the absence of caffeine, reduced phosphorylase a activity to 53% of controls at 1 mM Pi and to 85% at 5 mM Pi. At a 20-mM concentration, it was reduced to 27 and 57%, respectively (fig.1A, clear, broken line bars). When caffeine (1 mM) was added to 8 or 20 mM glucose, the inhibition was much greater (fig. 1A, stippled and solid bars). Thus, we have confirmed that the inhibitory effects of glucose and caffeine are synergistic. The synergistic effect of glucose and caffeine was essentially maximal at an 8 mM glucose concentration. The hepatic intracellular glucose concentration in a fasting animal is approximately 8 mM. Twenty mM represents the highest concentration likely to be present in a fed animal (Niewoehner and Nuttall, 1988) .

A physiological concentration of AMP (0.3 mM) (Boesiger et al., 1994) increased phosphorylase a activity ~ 1.9 fold at 1 mM Pi and ~ 1.3-fold at 5 mM Pi (fig.1B, clear, broken line bars). Caffeine at a 1 mM concentration decreased the stimulatory effect of the AMP (fig.1B, stippled and solid bars). Addition of glucose to the caffeine and AMP combination further reduced the AMP stimulatory effect in a concentration dependent manner. The inhibition at 20 mM glucose was 53% at 1 mM Pi, and 75% at 5 mM Pi, of the control activity. Thus, the inhibition was clearly less than that observed when only glucose and caffeine were added together (fig. 1A).

We previously reported that ADP inhibited phosphorylase a activity with an I_0.5 of ~3 mM at a 1 mM Pi concentration and the ADP inhibition was similar in the presence of AMP (Ercan et al., 1996). There also was little interaction between glucose and ADP. We wished to determine if ADP inhibition also was independent of caffeine and glucose. In our experiments when ADP, in concentrations from 1 to 4 mM, was added to AMP and caffeine (fig.1C, stippled and solid bars), the inhibition was similar to that observed in the absence of caffeine (fig.1C, clear, broken line bars).

When ADP at concentrations from 1 to 4 mM was added to the combination of caffeine (1 mM), AMP (0.3 mM) and glucose (8 mM) there was a progressive decrease in activity that was greater than when caffeine was absent (fig. 2A, stippled and solid bars vs. clear, broken line bars). This was particularly apparent at a 1 mM Pi concentration. At a 4-mM ADP concentration the activity was reduced to 30 and 50% of control activity at 1 and 5 mM Pi, respectively. When the glucose concentration was increased to 20 mM there was a further inhibition of activity (fig. 2B, stippled and solid bars). At a 4-mM ADP concentration the activity was reduced to 15 and 35% at 1 and 5 mM Pi, respectively. Thus, in this combination both ADP and glucose again inhibited phosphorylase a activity. The inhibition by ADP and glucose was independent of each other and was concentration dependent. The inhibition also was potentiated by caffeine.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Effect on phosphorylase a activity of varying ADP and ATP concentrations in the presence of a constant concentration of the other indicated effectors. The stippled bars represent activity at a 1 mM Pi concentration and the solid bars the activity at a 5 mM Pi concentration in the presence of caffeine. The clear, broken line bars indicate the activity in the absence of caffeine. (n=5 or greater for each).

As indicated previously, ATP within a physiological range inhibits phosphorylase a activity in a concentration-dependent manner and 0.3 mM AMP negates the inhibitory effect (Ercan et al., 1996). This also was observed in our studies. However, addition of 1.0 mM caffeine to ATP (6 mM) and AMP (0.3 mM) resulted in the phosphorylase a activity being essentially identical to that observed with AMP and caffeine in the absence of ATP, i.e., ATP no longer was an effector (figs. 2C vs. 1B).

ATP in concentrations varying from 2 to 8 mM resulted in a progressive, modest decrease in activity in the presence of caffeine (1 mM), AMP (0.3 mM) and glucose (8 and 20 mM) (figs. 2, D and E, respectively). The inhibitory effect of ATP was independent of the glucose inhibition. Overall, the inhibition by ATP was less than that when ADP was added to the caffeine (1 mM), glucose (8 and 20 mM) and AMP (0.3 mM) combination (figs. 2, A and B). Thus, the presence of glucose allowed the ATP inhibition to be expressed again, in the presence of AMP and caffeine.

Subsequently, we determined the phosphorylase a activity when caffeine was added to a combination of previously identified effectors (fig. 3A). The concentrations chosen were reported in vivo concentrations of ATP (6 mM), ADP (3 mM), AMP (0.3 m), fructose-1-P (0.3 mM), glucose-6-P (0.3 mM) and UDP-glucose (0.5 mM) (12). The latter two are only weak inhibitors of activity. Also, when added alone, fructose-1-P inhibits activity only at a 1.5 mM or greater concentration, a concentration that can be exceeded with fructose ingestion (Ercan et al., 1996, Niewoehner et al., 1984b.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   (A): Effect of phosphorylase a activity of varying glucose concentration in the presence of caffeine plus a combination of possible effectors at concentrations likely to be present in-vivo (AMP, ADP, ATP, glucose-6-P, UDP-glucose, fructose 1-P). (B) & (C): Effect on phosphorylase a activity of varying ADP concentration in the presence of the combinations of effectors at a glucose concentration of 8 mM (Combination 1), or 20 mM (Combination 2). (n=5 or greater for each).

In the absence of caffeine, we previously demonstrated that the phosphorylase a activity is the same with or without the addition of the combination of effectors. We also previously demonstrated that the addition of glucose in concentrations between 8 to 20 mM did not affect the phosphorylase a activity in the presence of the above combination of effectors (Ercan et al., 1996). In our experiments the addition of 1 mM caffeine resulted in a progressive decrease in activity as the glucose concentration was increased (fig. 3A). At a 20 mM glucose concentration, the activity at a 1 and 5 mM Pi concentration was only 18 and 30% of that in controls, respectively.

In the subsequent experiments, combination 1 refers to the combination of effectors plus 8 mM glucose. Combination 2 refers to the same combination of effectors and a 20-mM glucose concentration. To determine the interaction between effectors in the combination when a single effectors was removed, subtraction experiments were done.

When the ADP concentration in combination 1 was varied and caffeine was present at a 1 mM concentration, there was a progressive decrease in activity as the ADP concentration was increased (fig. 3B). This also was true at a 20 mM glucose concentration (combination 2) (fig. 3C).

When AMP was excluded the inhibition was greater, both when the glucose concentration was 8 mM (combination 1) (fig. 4A) and when the glucose concentration was 20 mM (combination 2) (fig. 4C). As expected, AMP antagonized the inhibition by glucose.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   (A & B): Effect on phosphorylase a activity when various effectors are removed from Combination 1. (C) & (D): Effects of removing various effectors from Combination 2 (See Figure 3 for more detail). (n=5 or greater for each).

When ADP was excluded from the mixture, the inhibition was less (figs.4, A and C). When ATP was excluded, the activity was only slightly increased compared to the complete mixture (figs. 4, A and C). Thus, ADP, was again a much more significant inhibitor than ATP when present in the combination of potential effectors. It also is clear that the weak inhibitors fructose-1-P, glucose-6-P and UDP-glucose had little effect on activity and they were not interacting with other effectors.

In the absence of both AMP and ADP the inhibitory effect was less than that with caffeine and glucose alone (figs. 4, B and D vs. 1, A). It was only slightly more than when only AMP was excluded. This suggests that ADP was opposing the effects of glucose and caffeine. Exclusion of AMP and ATP resulted in activity that was modestly greater than that observed with excluding AMP alone, i.e., the inhibitory effect was less than that observed with caffeine and glucose (figs. 4, B and D). This probably was due to a modifying effect of ADP on the inhibition by glucose and caffeine. When ADP and ATP were both excluded from the combination the inhibition was similar to that seen with caffeine + glucose + AMP (figs. 4, B and D vs. 1, B), i.e., there was little inhibition. When ADP, ATP and AMP were all excluded the expected caffeine and glucose effect was observed (figs. 4, B and D vs. 1, A). Overall, the data again indicate that fructose-1-P, glucose-6-P and UDP glucose at estimated in vivo concentrations do not significantly affect phosphorylase a activity in the presence of 1 mM caffeine.

Caffeine analogs. Adenosine at a 1 mM concentration had only a slight inhibitory effect at 1 mM Pi (~ 12%). It did not inhibit phosphorylase a activity at a 5 mM Pi concentration. When adenosine (1 mM) was added with AMP (0.3 mM) the stimulatory activity of AMP was reduced (table 1). This suggests they were competing for the same site. Glucose added at 8 or 20 mM reduced the phosphorylase a activity to the same level as without the addition of adenosine. That is, a synergistic effect was not present. The results were similar with theophylline.

	Discussion

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Caffeine (1,3,7-trimethylxanthine) (fig. 5) was reported as early as 1955 to inhibit skeletal muscle phosphorylase a. Both the a form and b form were inhibited and the inhibition was competitive with glucose-1-P. Also, the inhibitory effect was reversed by addition of AMP at concentrations as low as 10⁶ M. The 8-ethers and 8-thioethers of caffeine were found to be even more potent inhibitors of activity. Theophylline (1,3 dimethylxanthine), 1-methyltheobromine and the four possible trimethyl derivatives of uric acid were reported to be less active than caffeine. Adenine had little effect (Kihlman and Overgaard-Hansen, 1955) . 

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Structure of xanthine and the numbering system used. Caffeine has methyl groups at positions 1, 3 & 7. Theophylline has methyl groups at the 1 & 3 positions; theobromine at the 3 & 7 positions. In hypoxanthine the oxo group is absent at the 2 position. In azaxanthine a nitrogen replaces the carbon in the 8 position.

Subsequently, kinetic studies of both rabbit liver and muscle phosphorylase a demonstrated that caffeine and glucose inhibit the binding of the substrate, glucose-1-P, in a synergistic, competitive and nonexclusive manner (Kasvinsky et al., 1978). Caffeine was reported to bind preferentially to a nucleoside site on phosphorylase a. Using a rabbit liver phosphorylase a preparation, theophylline also was reported to inhibit activity but the K_i was twice as high for theophylline as for caffeine (0.8 vs. 0.4 mM). Allopurinol also inhibited activity in a competitive fashion. Other xanthine derivations were not studied (Kasvinsky et al., 1978) .

Using a rat liver phosphorylase a preparation, we have confirmed that caffeine strongly inhibits phosphorylase a activity and the inhibition is synergistic with glucose. A physiological concentration of AMP completely reversed the inhibition by caffeine. However, AMP which also completely reverses glucose inhibition (Ercan et al., 1996) reduced but did not eliminate the inhibitory effect of glucose when 1 mM caffeine was present. Caffeine and glucose have been reported to be competitive inhibitors of AMP binding to the enzyme although they bind to different sites (Kasvinsky et al., 1978). In our studies, because neither glucose or caffeine were present at a saturating concentration it is likely that the addition of caffeine diminished the binding of AMP and allowed a glucose- dependent inhibition of activity to occur at the physiological concentrations of glucose used. ADP, which also inhibits phosphorylase a activity (Ercan et al., 1996, Maddaiah and Madsen, 1966), continued to inhibit in the presence of caffeine and 0.3 mM AMP and glucose i.e., the inhibition was largely independent of these other effectors (fig. 1C and fig. 2, A and B). The ADP concentration in liver changes little in the absence of hypoxia. Thus, it is likely to be a constant and significant inhibitor in vivo.

ATP is an independent inhibitor (Ercan et al., 1996). It also is a competitive inhibitor of the activator AMP (Kasvinsky et al., 1978). In the present studies AMP eliminated ATP inhibition when both were present at a physiological concentration. Addition of caffeine, which itself is an inhibitor, attenuated the inhibitory effect of ATP on AMP activation (fig. 2C). In the presence of caffeine and AMP, addition of glucose now resulted in an inhibition by ATP. This was concentration dependent but was less than that expected from the addition of glucose alone. Thus, glucose but apparently not caffeine reduced the binding affinity of the enzyme for AMP when ATP was present (fig. 2, D and E).

These data indicate the potential complexity of the allosteric regulation of phosphorylase a in vivo. In any case, the presence of caffeine or a presumed physiological effector that mimics caffeine's effect in vivo could allow changes in glucose concentration to become an important regulator of phosphorylase a activity in vivo (fig. 3).

To probe the structural requirement for binding of xanthine derivatives to the caffeine binding site the effect of several analogs were determined in the absence and presence of other potential in vivo effectors. Theophylline (1,3-dimethylxanthine) did not independently affect activity at the concentration used in contrast to the striking inhibition by caffeine (1,3,7-trimethylxanthine) (table 1). A synergistic effect with glucose also was not present. It did reduce the stimulation of activity by AMP and as with caffeine it allowed an inhibitory effect of glucose to be seen in the presence of AMP. However, the inhibition was greater with caffeine as might be expected since caffeine but not theophylline has a synergistic effect with glucose (table 2).

Adenosine had affects that were very similar to those of theophylline; i.e., adenosine had little effect independently, but reduced AMP stimulation and did not synergize with glucose (tables 1 and 2). Adenosine concentrations in liver are much lower than used in our study (Sato and Ui, 1983). Thus, it is not likely to be a significant effector in vivo.

Xanthine itself and all of the other xanthine derivatives tested at a 1 mM concentration, strongly inhibited phosphorylase a activity and the inhibition was largely independent of any other effector (tables 1 and 2). Thus, neither xanthine nor any of these substituted derivatives mimicked the effect of caffeine's interaction with the other effectors.

Overall, the data indicate that xanthine and several of its derivatives are potent inhibitors. However, a trimethylated structure or a dimethylated structure with one of the methyl groups in the seven position is necessary for a synergistic interaction with glucose, at least at a 1 mM concentration. Unfortunately, theobromine (3,7-dimethylxanthine) was not tested. More detailed kinetic analysis of these inhibitors will be of interest in the future.