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 ac- tion 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 C 0 20 40 60 80 100 M0.40.26_ Minutes 0 FIG. 1. The effect of uridine on the UTP content of rat dia- phragm. Hemidiaphragms were incubated for various periods of time at 370 in the presence and absence of 5 mM uridine (gas-phase oxy- gen) 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.