Effect of Uridine on Cellular UTP and Glycogen Synthesis in Skeletal Muscle: Stimulation of UTP Formation by Insulin (Glycogen Formation/UTP Synthesis) ELLA S

Proc. Natl. Acad. Sci. USA Vol. 74, No. 6, pp. 2339-2342, June 1977 Biochemistry

Effect of uridine on cellular UTP and glycogen synthesis in skeletal muscle: Stimulation of UTP formation by insulin (glycogen formation/UTP synthesis) ELLA S. HAUGAARD, KATHERINE B. FRANTZ, AND NIELS HAUGAARD Department of Pharmacology, Medical School, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Communicated by George B. Koelle, March 23, 1977

ABSTRACT The relation between cellular uracil nucleo- termined on a torsion balance after gentle blotting on filter tides and ability to synthesize glycogen was studied in rat dia- paper. The results of metabolic measurements are expressed phragm incubated in vitro. In the absence of exogenous uridine the tissue content of UTP and rate of glycogen synthesis de- in units/g of wet weight. The tissues were incubated at 370 for creased with time. Uridine added to the medium increased various periods of time in 25-ml erlenmeyer flasks containing cellular UTP and UDPG and stimulated glycogen synthesis. 3.0 ml of medium. The standard incubation medium consisted Insulin significantly increased the synthesis of UTP from ex- of 40 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic tracellular uridine. This action of insulin appeared to be due to acid (Hepes) (pH 7.2)/5 mM MgCI2/5 mM KCI/105 mM a stimulation of phosphorylation of the nucleoside and not to NaCl/6 mM glucose. When uridine was added, the concen- an effect on transport at the concentrations of uridine studied. tration However, an effect of insulin on transport of uridine at low of NaCl was decreased to maintain isosmolarity. Insulin concentrations cannot be excluded. when present was at a concentration of 30 milliunits/ml. The flasks were gassed for 30 sec with 02 at the start of the incu- Studies of the regulation of glycogen synthesis in muscle have bation and again after 45 min of incubation. been concerned primarily with the properties of glycogen Rate of Glycogen Synthesis. The ability to synthesize gly- synthase (EC 2.4.1.11; UDP glucose:glycogen 4-a-glucosyl- cogen was determined by incubating the hemidiaphragms for transferase), the rate-limiting enzyme that catalyzes the reac- 10 min at 370 in 2.0 ml of the standard medium containing 6 tion: UDPG + (glucose),, = UDP + (glucose),,+,. This enzyme mM [U-14C]glucose (120,000 cpm/htmol) with or without 30 exists in two or more forms (1, 2), the best characterized being milliunits of insulin. The incorporation of isotope into glycogen designated as the independent (I or a) and dependent (D or b) was determined as described (5, 6). forms differing in their Ka values for activation by glucose-6-P Determination of UTP and UDPG. In experiments in which and in their apparent Km values for UDPG. Glycogen synthase these metabolites were determined, the tissues were ground in b is probably inactive physiologically since it is strongly in- chilled mortars with 5% trichloroacetic acid (1 ml/10-mg of hibited by tissue metabolites and its Km for UDPG is in the tissue). The trichloroacetic acid was removed by extraction with order of 0.2 mM, whereas glycogen synthase a is less sensitive ether. Samples of the extract were analyzed using a modifica- to inhibition and has a Km of about 0.06 mM (2). Since the action of the method of Keppler et al. (7). tual concentration of UDPG in muscle is approximately 0.03 Uptake of Uridine by Tissues. Hemidiaphragms were in- mM (3), it is clear that any changes in the cellular concentration cubated for various periods of time with different concentra- of this substrate would markedly alter the rate of glycogen tions of [U-14C]uridine (150,000 cpm/Amol). At the end of the synthesis. incubation the tissues were rinsed four times for 15 sec each in The formation of UDPG occurs by the reaction: UTP + chilled 0.15 M NaCl. They were blotted on filter paper after glucose-1-P = UDPG + P-P and is catalyzed by UDPG pyro- each rinsing and finally ground in 0.63 M perchloric acid (1 phosphorylase (EC 2.7.7.9; UTP:a-D-glucose-l-phosphate ml/100 mg). After centrifugation, an aliquot of the supernate uridylyltransferase), an enzyme that has been found not to be was neutralized with a mixture of triethanolamine and K2C03. rate limiting in the synthesis of glycogen in muscle (4). How- Radioactivity of samples of the neutralized extract was deter- ever, it seemed possible to us that the availability of UTP for mined in a Packard- Liquid Scintillation Counter. In experi- the synthesis of UDPG could be a limiting factor in glycogen ments in which hemidiaphragms had been incubated with 5 formation and hence the rate of synthesis of this polysaccharide mM radioactive uridine for 10 or 90 min, washout experiments could be influenced by the intracellular concentration of UTP. were done. Of the total radioactivity in the tissue present'after If this is true, additional supply of UDPG from exogenous uri- the preliminary washings, 66% represented uridine that had dine by the pyrimidine nucleotide salvage pathway should lead entered the intracellular space. For each of the experiments on to an increase in the rate of formation of glycogen. The ex- uptake of labeled uridine we, therefore, assumed that 66% of periments reported here were designed to study this prob- the measured radioactivity originated from intracellular uridine lem. and uracil derivatives. For the calculation of the intracellular concentration of uridine we used the value for intracellular MATERIALS AND METHODS water of 57.4% of tissue wet weight, as determined by the Materials. [U-14C]Glucose and [U-14C]uridine were ob- mannitol space method (8). tained from New England Nuclear, and uridine was from Calbiochem. The insulin preparation used was crystalline RESULTS porcine insulin (Eli Lilly and Co.; lot 615-DG3-10). The concentrations of UTP in the muscle were determined after Tissue Incubation. Male Wistar rats (125-150 g) fed freely incubation in the absence and presence of uridine in the me- were killed by decapitation and the diaphragms were removed. dium (Fig. 1). The level of UTP falls with time in the absence and collected in chilled 0.15 M NaCl. Wet weights were de- of uridine. However, in the presence of this nucleoside at a 2339 Downloaded by guest on September 26, 2021 2340 Biochemistry: Haugaard et al. Proc. Natl. Acad. Sci. USA 74 (1977)

250

+ 5 mM Uridine 200~ E 0+ In 0) .- 1.0 _ E 150.

c D 100 Control 0.8

50 c, 0.4 a

0 20 40 60 80 100 M0.40.26_ Minutes 0 FIG. 1. The effect of uridine on the UTP content of rat diaphragm. Hemidiaphragms were incubated for various periods of time at 370 in the presence and absence of 5 mM uridine (gas-phase oxygen) and the tissue content of UTP was determined. n = 4-8. 2 4 6 8 10 concentration of 5 mM there is a large increase in the cellular Uridine in medium, mM concentration of UTP. When hemidiaphragms were incubated with and without insulin in a medium containing 5 mM uracil FIG. 2. Effect of varying concentrations of uridine on the rate of (results not shown), the tissue content of UTP was the same as glycogen synthesis by rat diaphragm. Hemidiaphragms were incu- in controls even after 90 min. This indicates that uridine is bated for 90 min at 370 in media containing varying concentrations of uridine in the presence and absence of insulin. The tissues were converted to UTP and other uracil derivatives by the salvage blotted, transferred to media containing [14C]glucose, and reincubated pathway involving a direct phosphorylation as the initial for 10 min. The incorporation of isotope into glycogen was determined. step. Gas-phase oxygen was used in both incubations. n = 8-12. The rate of glycogen synthesis in vitro decreases with time of incubation. When hemidiaphragms are incubated with The UTP content of the hemidiaphragms at the end of the [U-"4C]glucose for 10 min without preincubation, the amount preincubation period was determined in parallel experiments of radioactive glucose incorporated into glycogen was found (Table 1). The synthesis of UTP from uridine occurs over a wide to be 1.56 i 0.14 ,Amol/g of tissue (N = 5). The rate of incor- range of uridine concentrations. In comparing the mean values poration of glucose into glycogen declined to 0.30 i 0.01 for UTP, we note that the effects of uridine on tissue UTP were Atmol/g of tissue per 10 min (n = 12) after 90 min of preincu- statistically significant at all concentrations of uridine in the bation. In order to determine whether the glycogen synthetic presence of insulin and at concentrations of 1 mM and above capacity of the muscle could be partially restored when the in the absence of hormone. When additional experiments were UTP content of the tissues is increased, we preincubated included and results were paired with respect to uridine, the hemidiaphragms for 90 rain in media containing varying effects of 0.05 and 0.5 mM uridine without insulin were also concentrations of uridine in the presence and absence of insulin. significant. Synthesis of UTP from uridine was increased by The tissues were then transferred to media containing ra- insulin. This action of the hormone was significant at all con- dioactive glucose and glycogen synthesis was determined centrations of uridine at and above 1 mM and at three different during a 10-min incubation in the absence of uridine. periods of incubation. The results reported in Fig. 2 show that the rate of glycogen The effect of insulin could be due either to increased trans- synthesis increases with the concentration of uridine in the port of uridine into the cell or to increased phosphorylation of medium, in the presence and absence of insulin, and that the the nucleoside. In order to determine whether insulin increased effect of the nucleoside is greater in the presence of the hor- the transport of uridine into the cell, we incubated hemidia- mone. phragms in media containing varying concentrations of [ U-

Table 1. Formation of UTP from uridine by rat diaphragm incubated in the presence and absence of insulin Time of Uridine in Insulin Significance of incubation, medium, UTP, nmol/g effect, insulin effect min mM n No insulin + Insulin nmol/g (paired differences)

90 0 8 71.4 ± 3.3 70.8 I 5.2 -0.6 ± 6.0 NS* 90 0.05 7 82.7 + 4.8 90.6 i 7.0 +7.9 + 8.3 NS* 90 0.5 7 88.0 ± 10.3 103.2 i 5.1 +15.2 ± 11.3 NS* 90 1.0 6 111.6 ± 13.3 142.4 i 9.2 +30.8 ± 9.5 P < 0.025 90 2.5 5 136.2 ± 11.8 167.2 ± 13.0 +31.0 ± 7.6 P < 0.025 90 5.0 4 190.6 + 7.5 222.5 i 7.5 +31.0 i 9.9 P < 0.05 30 5.0 8 148.5 + 3.3 162.0 i 3.3 +13.5 ± 4.3 P < 0.025 60 5.0 6 176.7 + 8.7 198.1 ±7.6 +21.4 ± 6.5 P <0.025 Paired hemidiaphragms were incubated with or without insulin for 30,60, or 90 min in media containing varying amounts of uridine and the UTP was determined. Values are ± SEM. * Not significant. Downloaded by guest on September 26, 2021 Biochemistry: Haugaard et al. Proc. Natl. Acad. Scd. USA 74 (1977) 2341

040 -

al0.35035-/ E

0 0.30 C E 0.6- 0.5mM n 025

0.4- C 0.20 C C0. ~,0.15- 0

*a 0.10 _ C 30 60 90 Minutes M 0.05 FIG. 3. Uptake of [14C]uridine by rat diaphragm in the presence C (0) and absence (0) of insulin. Hemidiaphragms were incubated at 40 80 120 160 200 370 in media containing different concentrations of [U-14C]uridine Increase in UTP, nmol/g (gas-phase oxygen) and the uptake of the radioactive uridine was FIG. 4. Relation between the increase in the UTP content and determined. the increase in rate of glycogen synthesis by rat diaphragm after incubation for 90 min at different concentrations of uridine. Data were 14C]uridine, with or without insulin, and the radioactivity of obtained from Fig. 2 and Table 1. Control value for glycogen synthesis the tissue was determined. The amounts of uridine and other after 90 min of incubation without uridine: 0.301 + 0.012 jimol of uracil metabolites present intracellularly were calculated as [14C]glucose incorporated into glycogen/g of tissue per 10 min without described in Materials and Methods. Results presented in Fig. insulin. The corresponding value with insulin was 0.580 + 0.022 3 show that uridine readily penetrates the muscle cell and gmol/g per 10 min (see Table 1 for UTP control values). (0) Without reaches a concentration in the cell close to that of the incubation insulin; (0) with insulin. no on of by the medium. Insulin has effect the uptake uridine points obtained from experiments with insulin fall on the same appears at the concentration mM) only tissue. It that highest (1 curve as the values from experiments carried out in the absence a that enters the cell is metabolized. For fraction of the uridine of hormone. It is evident that the greater effect of'uridine in example, after 90 min of incubation the increase in UTP is 40 increasing glycogen synthesis seen with insulin (see Fig. 2) is nmol/g in the absence of insulin and 72 nmol/g in the presence caused the hormonal stimulation of UTP synthesis. of the hormone (Table 1), whereas about 550 nmol/g of ra- by dioactive uracil derivatives, calculated as uridine, are present in the tissue. At lower concentrations of medium uridine (i.e., 0.05 and 0.02 mM) the total concentration of uracil derivatives DISCUSSION in the cell tends to become larger than medium uridine (see Fig. The experiments show that the cellular-concentration of UTP 3) and the proportion of tissue uridine metabolized in- can be a limiting factor in the ability of muscle to synthesize creases. glycogen. When rat hemidiaphragms are incubated in vitro The UDPG content of the tissues was determined after 90 there is a fall in UTP content and in the rate of glycogen syn- min of incubation in some of the experiments. In the absence thesis both in the absence and presence of insulin. When uridine of added uridine, the level of UDPG was 24.0 1.8 nmol/g (n is present in the medium, the tissue content of UTP increases = 16); in the presence of 5 mM uridine, the value was 42.1 + and this is associated with a much smaller fall in the rate of 3.5 nmol/g (n = 12). The effect of uridine was +18.1 ± 3.5 glycogen synthesis than in control tissues and a better mainte- nmol/g (P < 0.001). This 75% increase in UDPG corresponds nance of the ability of the muscle to respond to insulin. The to a 79% increase in the rate of glycogen synthesis produced by effect of uridine on the rate of glycogen synthesis seems to de- the same concentration of uridine (see Fig. 2). pend upon the conversion of part of the uridine into UTP and It is evident from the studies presented here that the rate of UDPG. glycogen synthesis in diaphragm muscle in vitro is markedly Uridine does not stimulate glycogen synthesis during a 10- dependent on the cellular content of UTP and the concomitant min period of incubation of fresh tissues with the nucleoside. change in the concentration of UDPG. Since insulin stimulates It has also been observed that the percent increase in glycogen the formation of UTP from uridine, it is obvious that the greater synthesis produced by a given concentration of uridine increases effect of uridine in increasing the rate of glycogen synthesis seen with time of incubation. in the presence of insulin (Fig. 2) could be due to the rise in UTP Most of the experiments were carried out with concentrations content produced by the hormone. That this appears to be true of uridine greater than those occurring physiologically. Plasma is illustrated in Fig. 4. concentrations of uridine + uracil in human beings have been To construct this graph we used data from the experiments estimated to be approximately 0.02 mM (9). However, uridine in which diaphragms were incubated for 90 min in the absence entered the cell during incubation with concentrations as low and in the presence of different concentrations of uridine. The as 0.02 and 0.05 mM, and at the latter concentration the UTP increase above controls without uridine in the rate of glycogen content of the tissue was higher than for controls. It should also synthesis during a subsequent 10-min period was plotted as a be considered that, in vivo, tissues are continuously exposed to function of the increase above control in UTP content of the uridine present in the extracellular fluid. tissue at the end of the 90-min preincubation. It is clearly seen The experiments of Rossi (10) demonstrate that the perfused that there is a linear increase in rate of glycogen synthesis as the rabbit heart can concentrate uridine from a perfusion medium UTP concentration increases over a wide range and that the containing 0.02 mM of this substance. The major portion of the Downloaded by guest on September 26, 2021 2342 Biochemistry: Haugaard et al. Proe. Natl. Acad. Sci. USA 74 (1977) uridine that enters the heart is phosphorylated and present in 1. Villar-Palasi, C. & Larner, J. (1960) "Insulin-mediated effect on the pool of soluble uracil nucleotides. In a recent paper Kypson the activity of UDPG-glycogen transglucosylase of muscle," and Hait (11) report that uridine stimulated the uptake of Biochim. Biophys. Acta 39, 171-173. 2. Roach, P. J., Takeda, Y. & Larner, J. (1976) "Rabbit skeletal glucose and the synthesis of glycogen in rat diaphragm in vitro. muscle glycogen synthase. I. Relationship between phosphoryl- These findings agree in general with our observations. However, ation state and kinetic properties," J. Biol. Chem. 251, 1913- the authors did not measure the UTP content of the tissue. 1919. The findings that the cellular concentrations of uracil nu- 3. Keppler, D., Rudigier, J. & Decker, K. (1970) "Enzymatic de- cleotides can be limiting in the synthesis of glycogen indicate termination of uracil nucleotides in tissue," Anal. Biochem. 38, that alterations in the metabolism of these substances can 105-114. markedly influence carbohydrate metabolism and the action 4. Villar-Palasi, C. & Larner, J. (1961) "Insulin treatment and in- of insulin. Is there, for example, a decrease in UTP in tissues creased UDPG-glycogen transglucosylase activity in muscle," from diabetic animals and does hypoxia produce a rapid fall Arch. Biochem. Biophys. 94,436-442. of synthesis of 5. Thomas, J. A., Schlender, K. K. & Larner, J. (1968) "A rapid filter in UTP with a concomitant decrease in the rate paper assay for UDP glucose-glycogen glucosyltransferase, in- glycogen? cluding an improved biosynthesis of UDP-14C-glucose," J. Anal. Probably the most important aspect of our study is the oh- Biochem. 25, 486-499. servation that insulin stimulates the synthesis of UTP in muscle. 6. Haugaard, E. S., Mickel, R. A. & Haugaard, N. (1974) "Actions Peck and Messinger (12) showed that insulin stimulated the of lithium ions and insulin on glucose utilization, glycogen syn- incorporation of labeled uridine into RNA and uracil nucleo- thesis and glycogen synthase in the isolated rat diaphragm," tides in isolated bone cells, and similar observations were made Biochem. Pharmacol. 23, 1675-1685. by Rillema (13) in experiments with mammary gland explants. 7. Keppler, D., Gawehn, K. & Decker, K. (1974) in Methods of The results of these experiments and our own observation that Enzymatic Analysis, ed. Bergmeyer, H. U. (Academic Press, UTP from uridine is increased by insulin indi- New York), Vol. 4, pp. 2172-2178. net synthesis of 8. Haugaard, E. S., Davidheiser, S. & Haugaard, N. (1976) "Effects cate that the hormone stimulates the initial phosphorylation of of epinephrine and cyclic AMP phosphodiesterase inhibitors on uridine. One mechanism of such an action of insulin could be the glycogen synthetic pathway and glucose content in skeletal a general increase in the availability of ATP for phosphorylative muscle," Biochem. Pharmacol. 25, 439-445. reactions. Experiments by Eboue-Bonis et al. (14) with rat di- 9. Parry, T. E. & Blackmore, J. A. (1974) "Serum 'uracil + uridine' aphragm in vitro have shown that the incorporation of inor- levels in normal subjects and their possible significance," J. Clin. ganic phosphate into high-energy compounds is markedly Pathol. 27, 789-793. stimulated by insulin in this tissue. Another possibility is that 10. Rossi, A. (1975) "Incorporation of uridine by the perfused rat uridine-cytidine kinase (EC 2.7.1.48; ATP:uridine 5'-phos- heart," Life Sci. 16, 1121-1132. photransferase), an enzyme rate limiting in the synthesis UTP 11. Kypson, J. & Hait, G. (1976) "Effects of uridine and inosine on could be under glucose metabolism in skeletal muscle and activated lipolysis in (15) and possibly existing in multiple forms (16), adipose tissue," J. Pharmacol. Exp. Ther. 199, 565-574. hormonal control and activated by insulin. 12. Peck, W. A. & Messinger, K. (1970) "Nucleoside and ribonucleic The action of insulin in increasing the cellular concentration acid metabolism in isolated bone cells," J. Biol. Chem. 245, of UTP in skeletal muscle from exogenous uridine provides a 2722-2729. mechanism by which the hormone can stimulate glycogen 13. Rillema, J. A. (1975) "Rapid effects of insulin on uridine me- synthesis that is additional to the well established activation of tabolism in mammary gland explants," Am. J. Physiol. 228, glycogen synthase (4). However, uracil nucleotides serve also 1531-1534. as intermediates in the formation of glycoproteins of cell 14. Eboue-Bonis, D., Chambaut, A. M., Volfin, P. & Clauser, H. membranes (17, 18) so that an effect of insulin on UTP synthesis (1963) "Action of insulin on the isolated rat diaphragm in the in of cellular membranes. presence of actinomysin D and puromycin," Nature 199, could lead to alterations the properties 1183-1184. If insulin also influences the metabolism of cytosine nucleotides, 15. Anderson, E. P. & Brockman, R. W. (1964) "Feedback inhibition such an action of the hormone could be involved in the effect of uridine kinase by cytidine triphosphate and uridine triphos- of insulin on the synthesis of phospholipids (19). Finally, phate," Biochim. Biophys. Acta 91, 380-386. changes in pyrimidine nucleotide metabolism produced by 16. Krystal, G. & Webb, T. (1971) "Multiple forms of uridine kinase insulin would be reflected in alterations of nucleic acid and in normal and neoplastic rat liver," Biochem. J. 124, 943-947. protein metabolism (20). 17. Levine, R. L., Hoogenraad, N. J. & Kretchmer, N. (1974) "A review: Biological and clinical aspects of pyrimidine metabolism," Pediat. Res. 8,724-734. This study was aided by a grant from Muscular Dystrophy Associ- 18. Krisman, C. R. & Barengo, R. (1975) "A precursor of glycogen ations of America, Inc. and by Research Grant HL-01813 from the biosynthesis: a-1,4-glucan-protein," Eur. J. Biochem. 52, National Heart, Lung and Blood Institute of the National Institutes of 117-123. Health, U.S. Public Health Service. 19. Stein, J. M. & Hales, C. N. (1974) "The effect of insulin on 32p The costs of publication of this article were defrayed in part by the incorporation into rat fat cell phospholipids," Biochim. Biophys. payment of page charges from funds made available to support the Acta 337, 41-49. research which is the subject of the article. This article must therefore 20. Wool, I. G., Stirewalt, W. S. & Moyer, A. N. (1968) "Effect of be hereby marked "advertisement" in accordance with 18 U. S. C. diabetes and insulin on nucleic acid metabolism of heart muscle," §1734 solely to indicate this fact. Am. J. Physiol. 214, 825-831. Downloaded by guest on September 26, 2021