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Incorporation of Glucose, Pyruvate, and Citrate Into Platelet Glycogen; Glycogen Synthetase and Fructose-1,6-Diphosphatase Activity

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Incorporation of Glucose, Pyruvate, and Citrate Into Platelet Glycogen; Glycogen Synthetase and Fructose-1,6-Diphosphatase Activity Glycogenesis and glyconeogenesis in human platelets: Incorporation of glucose, pyruvate, and citrate into platelet glycogen; glycogen synthetase and fructose-1,6-diphosphatase activity Simon Karpatkin, … , Arthur Charmatz, Richard M. Langer J Clin Invest. 1970;49(1):140-149. https://doi.org/10.1172/JCI106212. Washed human platelets are capable of depositing 1-4 as well as probable 1-6 glucosyl linkages onto preexistent glycogen primer. They are also capable of degrading (glycogenolysis) newly synthesized 1-4 as well as probable 1-6 glucosyl linkages. A higher rate of glycogen synthesis was found in platelet suspensions containing lower concentrations of platelets. This was shown to result from decreased glycogen degradation and consequent increased residual glycogen primer in low platelet suspensions. The increased glycogen content of low platelet suspensions was not a result of platelet washing, removal of platelets from plasma, or release of platelet metabolites into the media. The enzyme glycogen synthetase was found to be present at a rate of 5.2 μmoles of uridine diphosphate (UDP) glucose incorporated into glycogen per gram platelets per hour at 37°C. The Km for UDP glucose was 6.6 mmoles/liter. At optimum concentration of glucose 6-phosphate, the Km was reduced 4.6 fold and Vmax was increased 4.3-fold. Human platelets contain the glyconeogenic pathway. They incorporate pyruvate-14C and citrate-14C into platelet glycogen and contain an apparent fructose-1,6-diphosphatase. The apparent fructose-1,6-diphosphatase was activated by adenosine monophosphate (AMP) and adenosine diphosphate (ADP), inhibited by adenosine triphosphate (ATP), and shown to be rate limiting for glyconeogenesis at physiologic concentration of adenine nucleotide. Find the latest version: https://jci.me/106212/pdf Glycogenesis and Glyconeogenesis in Human Platelets INCORPORATION OF GLUCOSE, PYRUVATE, AND CITRATE INTO PLATELET GLYCOGEN; GLYCOGEN SYNTHETASE AND FRUCTOSE-1,6-DIPHOSPHATASE ACTIVITY SIMON KARPATKiN, AmTHuR CHARMATZ, and RIcmAuw M. LANGER From the Department of Medicine, New York University Medical Center, New York 10016 A B S T R A C T Washed human platelets are capable of stores, equivalent to those of skeletal muscle (1, 3). depositing 1-4 as well as probable 1-6 glucosyl link- Glycogenolysis in human platelets represents approxi- ages onto preexistent glycogen primer. They are also mately 50% of glycolytic flux through the Embden- capable of degrading (glycogenolysis) newly synthe- Meyerhof pathway in the presence of physiologic con- sized 1-4 as well as probable 1-6 glucosyl linkages. A centrations of glucose (1, 3). Human platelets contain higher rate of glycogen synthesis was found in plate- a glycogen degradative enzyme, phosphorylase, which let suspensions containing lower concentrations of plate- is similar in some respects to that of skeletal muscle lets. This was shown to result from decreased glycogen (4) but different in other respects (5). degradation and consequent increased residual glycogen This investigation was designed to determine whether primer in low platelet suspensions. The increased gly- glycogen synthesis takes place in human platelets and cogen content of low platelet suspensions was not a re- whether the "gluconeogenic" pathway was present, i.e., sult of platelet washing, removal of platelets from plasma, conversion of pyruvate and citrate to glucose or glyco- or release of platelet metabolites into the media. The gen. Isotopic labeling techniques were employed to mea- enzyme glycogen synthetase was found to be present sure incorporation of precursor into glycogen. The ex- at a rate of 5.2 /Amoles of uridine diphosphate (UDP) tent of glycogen labeling was determined by techniques glucose incorporated into glycogen per gram platelets which employed the isolation of previously labeled plate- per hour at 37'C. The Km for UDP glucose was 6.6 let glycogen during intervals of glycogen synthesis and mmoles/liter. At optimum concentration of glucose glycogen degradation. The isolated platelet glycogen was 6-phosphate, the Km was reduced 4.6 fold and V.a. was then analyzed for 1-4 glucosyl linkages by specific enzy- increased 4.3-fold. matic degradation. Human platelets contain the glyconeogenic pathway. They incorporate pyruvate-1'C and citrate-"C into plate- METHODS let glycogen and contain an apparent fructose-1,6-di- Fresh human platelets were obtained, washed, processed, phosphatase. The apparent fructose-1,6-diphosphatase and incubated at 370C as described previously (1, 3, 6). was activated by adenosine monophosphate (AMP) and All materials were obtained from vendors as described adenosine diphosphate (ADP), inhibited by adenosine previously (1, 3, 6). Uniformly labeled glucose-1'C, 261 mc/ and shown to be rate limiting for mmole, citrate-14C labeled in the 1-5 position, 1.84 mc/ triphosphate (ATP), mmole, and uniformly labeled uridine diphosphoglucose-1'C glyconeogenesis at physiologic concentration of adenine (UDPG), 204 mc/mmole were obtained from Nuclear- nucleotide. Chicago Corporation, Des Plaines, Ill. Uniformly labeled pyruvate-14C, 10.5 mc/mmole was obtained from International Chemical & Nuclear Corp., Burbank, Calif. 8-Amylase, INTRODUCTION phosphorylase, amylo-1,6-glucosidase, yeast phosphohexose isomerase, and fructose-1,6-diphosphate were obtained from Human platelets have a predominantly aerobic glycolytic Sigma Chemical Co., St. Louis, Mo. Fructose-1,6-diphos- metabolism (1, 2). They contain considerable glycogen phatase (1.1 U/mg) was obtained from Worthington Bio- chemical Corp., Freehold, N. J. Dr. Karpatkin is a Career Scientist of the Health Research Adenosine triphosphate (ATP), glucose, and lactate were Council of the City of New York (I-459). assayed enzymatically from neutralized perchloric acid ex- Received for publication 2 July 1969 and in revised form tracts, as described previously (1, 3). Fructose-1,6-diphos- 18 August 1969. phate was assayed by coupling the fructose-1,6-diphospha- 140 The Journal of Clinical Investigation Volume 49 1970 tase reaction to hexose isomerase and glucose 6-phosphate ,8-Amylase digestion of platelet glycogen. This procedure dehydrogenase and measuring the change in absorbance of was a minor modification of the method of Steinetz (8). triphosphopyridine nucleotide (NADP) to its reduced form, Briefly, 100 ,ug of f8-amylase was incubated with 1 mg of NAPDH, at 340 mg. The final concentration of reagents platelet glycogen in 1 ml of NaAc buffer, 16 mmoles/liter, employed were 0.46 mm NADP, 12.5 mm MgC12, 2 /g/ml pH 4.8 for 14 hr at 250C. These incubation conditions were glucose 6-phosphate dehydrogenase, 8 ,ug/ml hexose iso- found to be optimum. In control samples, ft-amylase was re- merase, 40 ,ug/ml fructose-1,6-diphosphatase, 20 mm mer- placed by a comparable volume of buffer. The glycogen captoethanol (MSH), in 50 mm Tris buffer, pH 7.5, and samples were then precipitated and redissolved. The dif- 200 ul/ml of neutralized perchlorate tissue extract. The ference in radioactivity and glycogen content between control reaction was started with addition of hexose isomerase. and enzyme digested samples represented the 1-4 linked tier Isotope experiments. Platelets were incubated in the pres- of branched glycogen beyond the 1-6 glucosyl linkage. The ehce of sufficient 1'C isotope to provide 1 X 106 cpm/ml in- limit dextrin obtained could be hydrolyzed to less than 5% cubation fluid. Various combinations of labeled and unlabeled of original radioactivity, as well as glycogen content, by in- glucose, pyruvate, or citrate were added to the incubation cubation with phosphorylase and amylo-1,6-glucosidase for media. After appropriate incubation, glycogen was pre- 20 hr at 250C. pared from platelets by extraction with 33%o KOH at 100°C Glycogen synthetase was measured by a radioisotopic pro- followed by two alcohol precipitations. Glycogen was as- cedure which was essentially a modification of the proce- sayed with anthrone reagent as described previously (3, 6). dure of Villar-Palasi and Larner (9). The platelet pellet Glycogen molarity is expressed as glucose units. The labeled was frozen in liquid nitrogen, ground in sand to a fine glycogen was dissolved in 1 ml of distilled water and an powder, and extracted in three times its volume of 50 mM aliquot pipetted into a glass counting vial containing Bray's Tris buffer, pH 8.2 containing 5 mM ethylenediaminetetraace- solution (7). Radioactivity was determined in a Beckman tate(EDTA) and 5 mm MSH. The optimal concentration LS100 scintillation spectrometer with isoset adjusted to give of MSH found to produce maximal enzyme activity was 5 96% counting efficiency. The radioactivity in the glycogen mmoles/liter. The extract was centrifuged at 4000 g at 40C samples was 60 times background after incubating plate- and the supernatant employed for glycogen synthetase de- lets with glucose-"C, 20 times background with pyruvate-14C, termination. The preparation was stable at 4°C for at least and 6-7 times background in citrate-"C experiments. The 1 wk. A 0.1 ml aliquot was added to 0.4 ml of enzyme incu- counting error was less than 3%. Control experiments re- bation solution at 37°C for 15 min containing (final con- vealed that addition of unlabeled platelet glycogen samples centration): UDPG-14C, 1 x 10. cpm/ml (varying concen- did not reduce the counting efficiency of a standard quantity tration of unlabeled UDPG), 0.5 g/100 ml glycogen, 5 mM of radioactivity. The concentration of tissue sample employed MSH, 5 mm EDTA, 50 mm Tris buffer, pH 8.2, and vary- for radioactivity measurements was well within the equal ing concentration of glucose-6-phosphate. The incubation was linear range of sample concentration addition vs. radio- terminated by the addition of 33% KOH, the glycogen activity obtained. extracted, and radioactivity assayed as described above. 1.3- 1.2 - 1.1- Z 0.9- o U0.8 (i 0.7- , 0.6- U 0 SUSPENSION 0 0.5- 0.4- 0.1 2 HOURS FIGURE 1 Effect of platelet concentration on incorporation of glucose-14C into platelet glycogen.
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