Limiting Factors for Glycogen Storage in Tumors I. Limiting Enzymes * VIJA! N. NIGAM, HELEN L. MACDONALD,t AND A. CANTERO (Montreal Cancer Institute, Research Laboratories, Notre Dame Ho8pital and UniveraW de Montreal, Montreal, Canada) SUMMARY The paper presents a study of various enzymes involved in the final accumulation of glycogen in tumor tissues. The solid tumors used are : Novikoff hepatoma, Walker 256 carcinosarcoma, Sarcoma 37, leukemia, and melanoma. It was observed that tumor tissues showed lower activity of phosphoglucomutase and glycogen synthetase as compared with that of normal liver and muscle. All tumors studied except melanoma also showed decreased UDPG' pyrophosphorylase levels. Activities for ATP- and UTP-regenerating enzymes—namely, pyruvate kinase and nucleoside disphospho kinase—in tumors were close to those found in normal tissues, thus providing sufficient quantities of cofactors (ATP and UTP) and energy for the polymerization process. UDPG was not diverted to UDPGA in tumors because of the absence of UDPG dehy drogenase. The lesion could be best described as a defective system for glycogen syn thesis, owing to low activities of the enzymes involved in the synthetic process (phos phoglucomutase and glycogen synthetase) which were unable to accomplish efficient transformation of G-6-P to G-1-P and of UDPG to glycogen, in the presence of com peting high rates of tumor glycolysis and normal polysaccharide degradation by phosphorylase. The decreased content of glycogen, together perimental work, designed to explain the defi with high rates of glycolysis, has been observed in ciency of glycogen in tumor. most tumor tissues and reported extensively (1, The metabolic pathway through which glucose 11). Although a number of reports have appeared is finally converted to glycogen begins with its (1, 10, 12, 40) to explain the high rate of lactic phosphorylation to glucose-6-phosphate through acid production in tumors, the reasons for the lack hexokinase and adenosine triphosphate; next, its of glycogen accumulation have not been fully ex transformation to glucose 1-phosphate by phos plored. In the light of the new pathway for glyco phoglucomutase; and, fiuially, conversion of the gen synthesis mediated through uridine diphos glucose 1-phosphate to uridine diphosphate glu phate glucose, the problem is open for further ex cose in the presence of uridine triphosphate and UDPG-pyrophosphorylase (14).' The UDPG 5This work was supported by grants from the National Cancer Institute of Canada. formed can transfer its glucose moiety to a pre Presented at the meeting of American Society of Biological formed glycogen molecule through glycogen syn Chemists in Atlantic City, April 10, 1961. thetase (16), or it can undergo dehydrogenation to t Technical assistant. UDP-glucuronic acid (35) and later transfer the ‘Theabbreviations used are: AMP, adenosine monophos glucuronic acid moiety to a suitable acceptor form phate; ADP, adenosine diphosphate; ATP, adenosinetriphos ing a glucuronide. phate; UDP, uridine diphosphate; UTP, uridine triphosphate; Thus glycogen accumulation requires systems UDPG, uridine diphosphate glucose; UDPGA, uridine diphos phate glucuronic acid; G-1-P, glucose-i-phosphate; G-6-P, for the regeneration of UTP and G-1-P, and their glucose-6-phosphate; DPN, diphosphopyridine nucleotide; subsequent conversion to UDPG by UDPG pyro TPN, triphosphopyridine nucleotide; GDP, guanosine diphos phosphorylase, and the presence of glycogen syn phate; CDP, cytidine diphosphate; IDP, inosine diphosphate. thetase to accomplish the transfer of glucose resi Received for publication May 22, 1961. dues of UDPG to an existent glucose polymer. 131 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1962 American Association for Cancer Research. 132 Cancer Research Vol. 22, February 1962 However, the presence of a synthesis system in BIocHEMIC@u@ PROCEDURES the tissue does not in itself provide for glycogen Glycogen synthetase was assayed according to storage, unless the glycogen-removing enzyme, Leloir and Goldemberg (16) in a system contain phosphorylase, is functioning at a normal or sub ing (in pmoles/0.115 ml), glycine buffer, pH 8.6, normal rate. 50; glucose-6-phosphate, pH 7, 0.825 ; UDPG, 0.5; This paper reports the activities of various en glycogen, 1 mg. ; and tissue homogenate, 0.01 ml. zymes responsible for the synthesis and degrada The digests were incubated at 87°C. for 10 mm tion of glycogen in tumor extracts, along with con utes and inactivated by boiling at 100° C. for 30 trol experiments on liver and muscle of both nor seconds, and the liberated UDP was estimated. mal and tumor-bearing animals. UDPG pyrophosphorylase digests consisted of MATERIALS AND METHODS (in @moles/3 ml), Tris-maleate buffer, pH 7.6, 100; UDPG, 1.25; magnesium chloride, 100; cysteine, CHEMICALS 1.59; TPN, 0.20; G-6-P dehydrogenase, 0.1 K The nucleotides (AMP, ADP, ATP, [JDP, units; phosphoglucomutase, 0.4 units; sodium py UDPG, DPN, TPN), sugar phosphates (G-1-P, rophosphate, 1.0; and 0.02 ml. of the supernatant G-6-P), G-6-P dehydrogenase Type IV, and pyru from a 10 per cent tissue homogenate. The control vate kinase were obtained from Sigma Chemical digest did not contain pyrophosphate. Equilibrium Co. Phosphopyruvate (tricyclohexyl amine salt) values were usually attained within 15—30minutes and myokinase were products of Mann Research of incubation at 37°C. The tumors, however, Laboratories, and rabbit liver glycogen of Nutri showed a lag period. The rise in extinction at 340 tional Biochemicals Corp. All other chemicals were m@ in the presence of TPN, phosphoglucomutase, the best reagent grade available from commercial and glucose-6-phosphate dehydrogenase was used sources. Crystalline phosphoglucomutase and as a measure of G-1-P concentration formed from adenylic acid deaminase were prepared according UDPG and pyrophosphate upon addition of the to Naj jar (24) and Kaickar (13), respectively. enzyme (22, 37). TRANSPLANTABLE MOUSE AND RAT TuMoRs Pi,ruvate kinase was measured by the system The studies reported here were carried out with employed for UDP or ADP estimation. The di the following tumors: Sarcoma 37 in white Swiss gests contained (in @imoles/0.5 ml) triethano mice, lymphatic leukemia in AKR mice, melanoma lamine buffer, pH 7.6, 30; phosphopyruvate, 0.9; in DBA/1J mice, Novikoff hepatoma in Sprague magnesium sulfate, 5 ; UDP or ADP, 0.1 ; and en Dawley rats, and Walker 256 carcinosarcoma in zyme supernatant, 0.05 ml. The pyruvate liberat Wistar rats. Normal mice and rats of the same ed was measured as a color complex with 2,4,- strain were used as control animals. dinitrophenyl hydrazine (16), after incubation at 370 C. for 15 mm. PREPARATION OF HOMOGENATES Nucleoside diphosphokinase was assayed ac AND SUPERNATANTS cording to the procedure of Berg and Joklik (4) The rats were stunned, decapitated, and bled, measuring ADP formation with myokinase and and the mice sacrificed by cervical dislocation. The adenylic acid deamiriase (23). livers, muscles, and tumors were quickly excised Phosphoglucomutase and UDPG dehydrogenase and placed in beakers surrounded by cracked ice. were assayed by procedures described by Najjar The tumors were freed as much as possible of ne (25) and Strominger et al. (36), respectively. crotic and connective tissue. Muscle was taken Phosphorylase activity was measured in the from the hind legs. Tissues were minced with scis presence and absence of 5'-AMP. The digests sors, and 10 per cent homogenates were prepared contained (in @imoles/ml), sodium citrate buffer, in ice-cold 0.25 M sucrose containing 0.001 M ver pH 5.9, 20; potassium fluoride, 46; G-1-P, pH 7.3, sene. Homogenization was carried out with a teflon 10; glycogen, 2 mg.; AMP, 0.04; 10 per cent ho pestle turning at about 600 r.p.m. for exactly 3 mogenate, 0.1 ml. The amount of inorganic phos minutes. The supernatant fluid was obtained by phate liberated was estimated according to Fiske centrifuging the tissue homogenates at 100,000 and Subbarow, with the presence or absence of Xg for 35 minutes at 00 C. A refrigerated Spin 5'-AMP used as a measure of the phosphorylase CO Model L centrifuge was used. About 1 gm. of a and b activities in the tissue extracts. tissue was added to hot KOH for glycogen esti Glycogen was isolated from hot KOH as de mation. scribed by Good, Kramer, and Somogyi (9) and, Glycogen synthetase and UDPG pyrophos after acid hydrolysis, estimated as glucose by the phorylase in the homogenate were assayed the Nelson (26) method.. same day. The remaining enzyme solutions were For the determination of glucose and oligo divided in tubes and frozen at —20°C. saccharides the tissue was extracted with 70 per Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1962 American Association for Cancer Research. NIGAM et al.—Limiting Enzymes and Glycogen Storage 133 cent ethanol for 24 hr. at 0°—5°C.,centrifuged, of glucosyl oligosaceharides in the liver has been concentrated, and chromatographed. The individ shown by Fishman and Sie (7) and their origin ual sugars were eluted from the paper and esti traced by Olavarria (29) as owing to the presence mated as glucose, as described by Nigam and of enzymes in liver which make labeled maltose, Gin (27). maltotriose, maltotetraose, and other homologs RESULTS from glycogen-C'4. A rapid decrease in the con Glycogen content of tumors and of the liver and centration of the oligosaccharides during fasting muscle of normal and tumor-bearing animak.—The has also been reported (32). A situation similar to low content of glycogen in tumors has been re fasting occurred during the growth of the liver ported by LePage (18) and others (2, 10). In the tumor. Six days after the implantation of the case of hepatoma, it has been shown that tumor tumor the animal lost its store of glycogen, with formation brings a subsequent decrease in the a subsequent decrease in the oligosaccharides.