Europ.J.clin.Invest. 5, 7-14 (1975)
Insignificance of Gluconeogenesis in Human Blood Platelets J. Schrijver, 3 , F. Koster, and W.C. Hiilsmann Department of Biochemistry I, Faculty of Medicine, Erasmus University, Rotterdam, The Netherlands Received: February 26, 1974, and in revised form: May 16, 1974
Abstract. Human blood platelets contain no detectable activity of the enzymes fructose diphosphatase (EC 3.1.3.11), phospho-enolpyruvate carboxykinase (EC 4.1.1.32) and pyruvate carboxylase (EC 6.4.1.1.). Glucose-6-phosphatase (EC 3.1.3.9) activity is very low. Phosphofructokinase present in human blood platelets, catalyzes a reaction which can be stimulated by AMP in a platelet homogenate, due to the presence of endogenous ADP and myokinase. These enzymes are responsible for the formation of fructose-6-phosphate from fructose-], 6-diphosphate. Pyruvate kinase (EC 2.7.1.40) in human blood platelets belongs to the M-type, which is not inhibited by A m , at least not under the conditions applied. The results obtained indicate that gluconeogenesis in human blood platelets is not present in the way which has been established for liver and kidney. Key words: Human blood platelets, gluconeogenesis, inborn errors of metabolism.
Abbreviations used:
demands all of the enzymes mentioned. Although the exact regulation mechanism by which pyruvate and lactate are converted to glucose is not known l i p , FDPase D-fructose-I, 6-diphosphate I-phosthe presence and importance of gluconeogenesis in phohydrolase (EC 3.1.3.11) liver and kidney is very well established 3 . PEPCK GTP: oxaloacetate carboxy-lyase (transIn order to be able to use human blood platelets phosphorylating) (EC 4 . 1 . 1 . 3 2 ) as an enzyme source for the diagnosis of metabolic PC Pyruvate:COp ligase (ADP) (EC 6.4.1.1) disorders, especially those concerned with the meta G6Pase D-glucose-6-phosphate phosphohydrolase bolism of pyruvate, we investigated whether throm(EC 3.1.3.9) bocytes are capable of synthesizing glucoses from PK ATP: pyruvate phosphotransferase (EC 2. pyruvate. Some reports on carbohydrate synthesis in 7.1.40) human blood platelets have appeared. Karpatkin e t PEP Phospho-enolpyruvate at. measured an incorporation of pyruvate and ciDTT Dithiotreito1 trate into endogenous glycogen of human blood TPP Thiamin pyrophosphate also demonstrated a platelets. Nakamura e t aZ. PGI D-glucose-I, 6-diphosphate:D-glucose-lphosphate phosphotransferase (EC 2.7.5.1) synthesis of glucose from pyruvate in intact G6PDH platelets with the aid of high concentrations of D-glucose-6-phosphate:NADP oxidoreductase (EC 1.1.1.49) added ATP and pyruvate. To our knowledge there is 6-phospho-D-gluconate:NAD(P) oxidoreduc- no direct evidence for the presence of human blood 6PGDH tase (EC 1.1.1.43) platelets of a number of enzymes unique to gluconeoPDH Pyruvate:lipoate oxidoreductase (acceptor--genesis. Therefore we tried to measure the activiacetylating) (EC 1.2.4.1) ties of these enzymes in the platelets. According Glc-6-P glucose-6-phosphate to the literature G6Pase is present 6 , whereas Fru-1,6-P~ fructose-], 6-diphosphate Karpatkin e t a l . described an apparent FDPase which is stimulated by AMP rather than inhibited, as reported for other tissues Furthermore, the properties of PK in human blood platelets have been investi.gated, because it is known that cells with Whether gluconeogenesis can occur in a cell or active gluconeogenesis contain a special type of not, depends on the presence of'the key enzymes PK (L-type), whose activity can be modulated alPEPCK, PC, FDPase and G6Pase. Synthesis of glucose losterically *-I4. The L-type PK is involved in the from glycerol requires FDPase and G6Pase, besides regulation of glycolysis and the enzyme can be a number of enzymes from the glycolytic pathway. switched off during gluconeogenesis. The conversion of pyruvate and lactate to glucose
'.
8
J. Schrijver e t aZ.: Gluconeogenesis in Human Blood Platelets
100 mM Tris-HC1 (BH 8.0), 10 mM MgC12, 20 mM KC1, 2 mM DTT, 5mM NAD , 2 mM TPP and 1 mM coenzyme A , variable concentrations of l-I4C pyruvate, 18 mM 1 . Isolation of human blood platelets carnitine and 20 pg of carnitine acetyltransferase (EC 2.3.1.7). Carnitine and carnitine acetyltransFresh human blood platelets were used for each ferase were added to convert the acetyl-CoA formed experiment and isolated by the following method. to acetylcarnitine to rule out a possible inhibition 50 ml of human blood was taken from normal adults of the PDH activity by acetyl-CoA. Control incuby venipuncture and mixed directly with 2 ml of a solution containing 0.8 X NaCl (w/v) and 0.67M EDTA bations were done in the absence of added coenzyme A and in the presence of 1 mM NaAs02, with and (w/v) as anticoagulant. After standing in a waterbath at 22OC for 30 m i n . , the blood was centrifuged without platelet homogenate. for 20 min. at room temperature in plastic tubes The activities of PC and PEPCK were measured in the direction of glucose synthesis: that is the conat 175 x g . The platelets were separated from the plasma proteins by passing 15 to 20 ml of the super- version of pyruvate to oxaloacetate catalyzed by natant at room temperature through a Sepharose 2B PC and the decarboxylation of oxaloacetate to PEP column (2.5 cm, X 25 cm), in accordance with the catalyzed by PEPCK. PEPCK was assayed at 3OoC in method of Tangen e t aZ. l 5 . a medium with 100 mM Tris-HCI (pH 8), 0.6 mM MnC12, For enzyme assays, the platelets separated by 13 mM MgS04, 7 mM KC1 and 7 mM KF, 4.7 mM ITP, gel filtration were centrifuged at O°C for 15 min. 2 mM oxaloacetate and 2 mM GSH in a total volume at 3 000 x g. The platelet pellet was suspended in qf 1.5 ml. PEP was measured by the method as given 2 ml of a solution which contained 10 mM Tris-HC1 by Czok and Lambrechts30. The formation of oxa(pH 7.4), 2 mM EDTA and 2 mM GSH or DTT. The ratloacetate from pyruvate and HI4C03 at 3OoC, cataliver homogenate was also made in this medium. When lyzed by the enzyme PC, was measured by converting PK was assayed EDTA and GSH or DTT were omitted. In the oxaloacetate produced to citrate with acetylthe determination of the activity of FDPase, the CoA and citrate synthase (EC 4.1.3.7) because of SH-compound was also omitted. The platelets were the instability of the reaction product. The redisintegrated either by freeze-thawing or by ultra- action medium (0.5 ml) contained: 100 mM Tris-HC1 sound (2 min. at 20,000 Hz, amplitude 7.5 p ) with (pH 7.4), 20 mM KCl, 10 1124 MgC12, 1 mM coenzyme the aid of an MSE Ultrasonic Disintegrator proA, 3 mM ATP, 33 mM NaH14C03, 6 mM Li-acetylphosvided with a titanium exponential microprobe. The phate, 0.25 X (w/v> bovine serum albumin, 12 mM platelet homogenate was centrifuged at O°C for pyruvate, 2 mM pyruvate, 2 IIM GSB, 25 pg of phos15 min. at 25,000 x g ; the supernatant (about 1.8ml) photransacetylase (EC 2.3.1.8) and 5 p g of citrate was used for the enzyme assays. When PDH was assynthase. sayed, the last step was omitted. The activity of pyruvate kinase was measured at pH 7.5 as described by Valentine e t a1.l6, except that 250 mM Tris-HC1 was used as buffer. 2. Incubations and enzyme assays The hydrolysis of Fru-I, 6;P2 by the platelet Oxidation and incorporation of I-l4C pyruvate: homogenate was measured at 37 C as the formation in glucose were measured by incubation in a waterof NADPH by coupling the reaction to phosphohexose bath at 37OC (shaken at 100 cycles/min.). Thromisomerase (PGI), G6PDH or by determining the reacbocytes were added to 1 ml of a Krebs Ringer phostion products P. and Fru-6-P and Glc-6-P; the last phate solution (pH 7.4) in a siliconised closed product results’from the presence of PGI in the glass vial, provided with a small central vessel, platelet homogenate. The formation of NADPH was which contained 200 p1 of a 1 M hyamine-hydroxide continuously recorded as an increase of the ab10-X solution in methanol, used for the fixation of sorbance at 340 nm in a standard reaction medium the liberated 14C02. The incubations were stopped containing: 50 mM Tris-HC1 (pH 7.4), 1 mM EDTA, by the addition of 100 p 1 of a 30 Z (w/v) solution 6 mM MgC12, 2.5 mM monoiodoacetamide to inhibit o f HCIOI, and after 30 min. the reaction medium was glyceraldehyde-3-pkosphate dehydrogenase (EC 1.2. neutralized with a solution of 1 M K2HPO4 and cenl.l2), 0.5 mM NADP , 10 pg of PGI, 10 pg of G6PDH trifuged to remove the precipitated KC104. 400 p 1 and 10 pg of 6PGDH in a total volume of 2 ml. When of the supernatant, which contained pyruvate, P. and the 9exose monophosphate esters were deter1 platelet glycogen and glucose, was put on a column mned, NADP and the coupling enzymes were omitted. (6m, X 8 cm) of well-washed Amberlite IRA 410 Inorganic phosphate (P.) was determined in accordawe resin, and eluted with distilled water to separate with the method of Fis%e and SubbaRow17 and Fru-6-P glucose pzus glycogen from pyruvate in the reaction and Glc-6-P at 340 nm in a reaction medium as used medium. Pyruvate was completely retained on the for the coupled FDPase assay. To get some inforcolumn and glucose and glycogen were eluted in the mation on the activities of G6Pase and the unspecivoid volume. The first 5 ml was collected in a fic phosphatases in the platelet homogenate, the was counting vial, which contained 10 ml of Instagel and hydrolysis of Glc-6-P and of 8-glycerophosphate . counted in a Packard Liquid Scintillation Spectropho-measured at 3OoC as a release of glucose and of P. tometer, model 544, with a counting error of less in a standard medium of 0.4 ml containing 75 mM than 3 %. buffer (citrate, Tris-HC1 or glycine-NaOH) and 50 The activity of PDH in a five times frozen and mM Glc-6-P or B-glycerophosphate. Glucose was dethawed platelet suspension, was measured during termined by the method described by Werner e t aZ.l8,. The G6Pase reactions with patients materials were 60 min. as the evolution 0f’~C02 from I-l4C PYruvate at 3OoC in a medium of 0.5 ml containing: performed in the citrate buffer (pH 6.5).
Materials and Methods
1-w
J. Schrijver e t a l . : Gluconeogenesis in Human Blood Platelets
Glycogen was determined with the method described by Huijing19. S'-Nucleotidase was obtained from Sigma Chemical Company. The other enzymes and PEP, ADP, AMP, ATP and hexosephosphate esters were obtained from Boehringer (Mannheim, Germany). 1-14C pyruvate was supplied by the Radiochemical Centre, Amersham, Bucks., England. All other reagents were of analytical grade. In all enzymatic assays the velocity rates were proportional with time and amount of protein.
9
Table 1 . The activities of PEPCK and PC in human blood platelets and in rat-liver. For measurements of the activities see Materials and Methods Source of homogenate
PEPCK~)
n
Human blood platelets
4
Rat-1iver
2
PCb)
Insignificance of Gluconeogenesis in Human Blood Platelets J. Schrijver, 3 , F. Koster, and W.C. Hiilsmann Department of Biochemistry I, Faculty of Medicine, Erasmus University, Rotterdam, The Netherlands Received: February 26, 1974, and in revised form: May 16, 1974
Abstract. Human blood platelets contain no detectable activity of the enzymes fructose diphosphatase (EC 3.1.3.11), phospho-enolpyruvate carboxykinase (EC 4.1.1.32) and pyruvate carboxylase (EC 6.4.1.1.). Glucose-6-phosphatase (EC 3.1.3.9) activity is very low. Phosphofructokinase present in human blood platelets, catalyzes a reaction which can be stimulated by AMP in a platelet homogenate, due to the presence of endogenous ADP and myokinase. These enzymes are responsible for the formation of fructose-6-phosphate from fructose-], 6-diphosphate. Pyruvate kinase (EC 2.7.1.40) in human blood platelets belongs to the M-type, which is not inhibited by A m , at least not under the conditions applied. The results obtained indicate that gluconeogenesis in human blood platelets is not present in the way which has been established for liver and kidney. Key words: Human blood platelets, gluconeogenesis, inborn errors of metabolism.
Abbreviations used:
demands all of the enzymes mentioned. Although the exact regulation mechanism by which pyruvate and lactate are converted to glucose is not known l i p , FDPase D-fructose-I, 6-diphosphate I-phosthe presence and importance of gluconeogenesis in phohydrolase (EC 3.1.3.11) liver and kidney is very well established 3 . PEPCK GTP: oxaloacetate carboxy-lyase (transIn order to be able to use human blood platelets phosphorylating) (EC 4 . 1 . 1 . 3 2 ) as an enzyme source for the diagnosis of metabolic PC Pyruvate:COp ligase (ADP) (EC 6.4.1.1) disorders, especially those concerned with the meta G6Pase D-glucose-6-phosphate phosphohydrolase bolism of pyruvate, we investigated whether throm(EC 3.1.3.9) bocytes are capable of synthesizing glucoses from PK ATP: pyruvate phosphotransferase (EC 2. pyruvate. Some reports on carbohydrate synthesis in 7.1.40) human blood platelets have appeared. Karpatkin e t PEP Phospho-enolpyruvate at. measured an incorporation of pyruvate and ciDTT Dithiotreito1 trate into endogenous glycogen of human blood TPP Thiamin pyrophosphate also demonstrated a platelets. Nakamura e t aZ. PGI D-glucose-I, 6-diphosphate:D-glucose-lphosphate phosphotransferase (EC 2.7.5.1) synthesis of glucose from pyruvate in intact G6PDH platelets with the aid of high concentrations of D-glucose-6-phosphate:NADP oxidoreductase (EC 1.1.1.49) added ATP and pyruvate. To our knowledge there is 6-phospho-D-gluconate:NAD(P) oxidoreduc- no direct evidence for the presence of human blood 6PGDH tase (EC 1.1.1.43) platelets of a number of enzymes unique to gluconeoPDH Pyruvate:lipoate oxidoreductase (acceptor--genesis. Therefore we tried to measure the activiacetylating) (EC 1.2.4.1) ties of these enzymes in the platelets. According Glc-6-P glucose-6-phosphate to the literature G6Pase is present 6 , whereas Fru-1,6-P~ fructose-], 6-diphosphate Karpatkin e t a l . described an apparent FDPase which is stimulated by AMP rather than inhibited, as reported for other tissues Furthermore, the properties of PK in human blood platelets have been investi.gated, because it is known that cells with Whether gluconeogenesis can occur in a cell or active gluconeogenesis contain a special type of not, depends on the presence of'the key enzymes PK (L-type), whose activity can be modulated alPEPCK, PC, FDPase and G6Pase. Synthesis of glucose losterically *-I4. The L-type PK is involved in the from glycerol requires FDPase and G6Pase, besides regulation of glycolysis and the enzyme can be a number of enzymes from the glycolytic pathway. switched off during gluconeogenesis. The conversion of pyruvate and lactate to glucose
'.
8
J. Schrijver e t aZ.: Gluconeogenesis in Human Blood Platelets
100 mM Tris-HC1 (BH 8.0), 10 mM MgC12, 20 mM KC1, 2 mM DTT, 5mM NAD , 2 mM TPP and 1 mM coenzyme A , variable concentrations of l-I4C pyruvate, 18 mM 1 . Isolation of human blood platelets carnitine and 20 pg of carnitine acetyltransferase (EC 2.3.1.7). Carnitine and carnitine acetyltransFresh human blood platelets were used for each ferase were added to convert the acetyl-CoA formed experiment and isolated by the following method. to acetylcarnitine to rule out a possible inhibition 50 ml of human blood was taken from normal adults of the PDH activity by acetyl-CoA. Control incuby venipuncture and mixed directly with 2 ml of a solution containing 0.8 X NaCl (w/v) and 0.67M EDTA bations were done in the absence of added coenzyme A and in the presence of 1 mM NaAs02, with and (w/v) as anticoagulant. After standing in a waterbath at 22OC for 30 m i n . , the blood was centrifuged without platelet homogenate. for 20 min. at room temperature in plastic tubes The activities of PC and PEPCK were measured in the direction of glucose synthesis: that is the conat 175 x g . The platelets were separated from the plasma proteins by passing 15 to 20 ml of the super- version of pyruvate to oxaloacetate catalyzed by natant at room temperature through a Sepharose 2B PC and the decarboxylation of oxaloacetate to PEP column (2.5 cm, X 25 cm), in accordance with the catalyzed by PEPCK. PEPCK was assayed at 3OoC in method of Tangen e t aZ. l 5 . a medium with 100 mM Tris-HCI (pH 8), 0.6 mM MnC12, For enzyme assays, the platelets separated by 13 mM MgS04, 7 mM KC1 and 7 mM KF, 4.7 mM ITP, gel filtration were centrifuged at O°C for 15 min. 2 mM oxaloacetate and 2 mM GSH in a total volume at 3 000 x g. The platelet pellet was suspended in qf 1.5 ml. PEP was measured by the method as given 2 ml of a solution which contained 10 mM Tris-HC1 by Czok and Lambrechts30. The formation of oxa(pH 7.4), 2 mM EDTA and 2 mM GSH or DTT. The ratloacetate from pyruvate and HI4C03 at 3OoC, cataliver homogenate was also made in this medium. When lyzed by the enzyme PC, was measured by converting PK was assayed EDTA and GSH or DTT were omitted. In the oxaloacetate produced to citrate with acetylthe determination of the activity of FDPase, the CoA and citrate synthase (EC 4.1.3.7) because of SH-compound was also omitted. The platelets were the instability of the reaction product. The redisintegrated either by freeze-thawing or by ultra- action medium (0.5 ml) contained: 100 mM Tris-HC1 sound (2 min. at 20,000 Hz, amplitude 7.5 p ) with (pH 7.4), 20 mM KCl, 10 1124 MgC12, 1 mM coenzyme the aid of an MSE Ultrasonic Disintegrator proA, 3 mM ATP, 33 mM NaH14C03, 6 mM Li-acetylphosvided with a titanium exponential microprobe. The phate, 0.25 X (w/v> bovine serum albumin, 12 mM platelet homogenate was centrifuged at O°C for pyruvate, 2 mM pyruvate, 2 IIM GSB, 25 pg of phos15 min. at 25,000 x g ; the supernatant (about 1.8ml) photransacetylase (EC 2.3.1.8) and 5 p g of citrate was used for the enzyme assays. When PDH was assynthase. sayed, the last step was omitted. The activity of pyruvate kinase was measured at pH 7.5 as described by Valentine e t a1.l6, except that 250 mM Tris-HC1 was used as buffer. 2. Incubations and enzyme assays The hydrolysis of Fru-I, 6;P2 by the platelet Oxidation and incorporation of I-l4C pyruvate: homogenate was measured at 37 C as the formation in glucose were measured by incubation in a waterof NADPH by coupling the reaction to phosphohexose bath at 37OC (shaken at 100 cycles/min.). Thromisomerase (PGI), G6PDH or by determining the reacbocytes were added to 1 ml of a Krebs Ringer phostion products P. and Fru-6-P and Glc-6-P; the last phate solution (pH 7.4) in a siliconised closed product results’from the presence of PGI in the glass vial, provided with a small central vessel, platelet homogenate. The formation of NADPH was which contained 200 p1 of a 1 M hyamine-hydroxide continuously recorded as an increase of the ab10-X solution in methanol, used for the fixation of sorbance at 340 nm in a standard reaction medium the liberated 14C02. The incubations were stopped containing: 50 mM Tris-HC1 (pH 7.4), 1 mM EDTA, by the addition of 100 p 1 of a 30 Z (w/v) solution 6 mM MgC12, 2.5 mM monoiodoacetamide to inhibit o f HCIOI, and after 30 min. the reaction medium was glyceraldehyde-3-pkosphate dehydrogenase (EC 1.2. neutralized with a solution of 1 M K2HPO4 and cenl.l2), 0.5 mM NADP , 10 pg of PGI, 10 pg of G6PDH trifuged to remove the precipitated KC104. 400 p 1 and 10 pg of 6PGDH in a total volume of 2 ml. When of the supernatant, which contained pyruvate, P. and the 9exose monophosphate esters were deter1 platelet glycogen and glucose, was put on a column mned, NADP and the coupling enzymes were omitted. (6m, X 8 cm) of well-washed Amberlite IRA 410 Inorganic phosphate (P.) was determined in accordawe resin, and eluted with distilled water to separate with the method of Fis%e and SubbaRow17 and Fru-6-P glucose pzus glycogen from pyruvate in the reaction and Glc-6-P at 340 nm in a reaction medium as used medium. Pyruvate was completely retained on the for the coupled FDPase assay. To get some inforcolumn and glucose and glycogen were eluted in the mation on the activities of G6Pase and the unspecivoid volume. The first 5 ml was collected in a fic phosphatases in the platelet homogenate, the was counting vial, which contained 10 ml of Instagel and hydrolysis of Glc-6-P and of 8-glycerophosphate . counted in a Packard Liquid Scintillation Spectropho-measured at 3OoC as a release of glucose and of P. tometer, model 544, with a counting error of less in a standard medium of 0.4 ml containing 75 mM than 3 %. buffer (citrate, Tris-HC1 or glycine-NaOH) and 50 The activity of PDH in a five times frozen and mM Glc-6-P or B-glycerophosphate. Glucose was dethawed platelet suspension, was measured during termined by the method described by Werner e t aZ.l8,. The G6Pase reactions with patients materials were 60 min. as the evolution 0f’~C02 from I-l4C PYruvate at 3OoC in a medium of 0.5 ml containing: performed in the citrate buffer (pH 6.5).
Materials and Methods
1-w
J. Schrijver e t a l . : Gluconeogenesis in Human Blood Platelets
Glycogen was determined with the method described by Huijing19. S'-Nucleotidase was obtained from Sigma Chemical Company. The other enzymes and PEP, ADP, AMP, ATP and hexosephosphate esters were obtained from Boehringer (Mannheim, Germany). 1-14C pyruvate was supplied by the Radiochemical Centre, Amersham, Bucks., England. All other reagents were of analytical grade. In all enzymatic assays the velocity rates were proportional with time and amount of protein.
9
Table 1 . The activities of PEPCK and PC in human blood platelets and in rat-liver. For measurements of the activities see Materials and Methods Source of homogenate
PEPCK~)
n
Human blood platelets
4
Rat-1iver
2
PCb)
