Inhibition by Glucose 6-Phosphate

Home , Phosphofructokinase

Proc. Nati. Acad. Sci. USA Vol. 82, pp. 1552-1554, March 1985 Medical Sciences High glucose concentrations partially release hexokinase from inhibition by glucose 6-phosphate (diabetes/kinetics) SHINYA FumII AND ERNEST BEUTLER* Third Department of Internal Medicine, Yamaguchi University School of Medicine, 1144 Kogushi, Ube, Yamaguchi, 755, Japan; and Scripps Clinic and Research Foundation, Department of Basic and Clinical Research, 10666 North Torrey Pines Road, La Jolla, CA 92037 Contributed by Ernest Beutler, November 2, 1984

ABSTRACT The phosphorylation of glucose by human cose concentrations, and although previous reports showed erythrocyte hexokinase follows classical Michaelis-Menten ki- no competition of these inhibitors with glucose, no details netics; hexokinase manifests maximum activity at 5 mM glu- were presented. In the present studies, we extensively ex- cose, and no further increase in activity can be measured at amined the velocity of the hexokinase reaction in the pres- higher glucose concentrations. However, the erythrocytes of ence of inhibitors and high concentrations of glucose in or- diabetics and normal erythrocytes incubated with high concen- der to shed light on the cause of the elevation of glucose 6- trations of glucose contain increased concentrations of glucose phosphate levels in the erythrocytes of diabetics. 6-phosphate. To elucidate the mechanism of accumulation of glucose 6-phosphate when erythrocytes are exposed to high glucose concentrations, hexokinase activity was examined in EXPERIMENTAL PROCEDURES the presence of naturally occurring inhibitors, such as glucose Partial Purification of Hexinas from Human Erythro- 1,6-bisphosphate, 2,3-diphosphoglycerate, ADP, and glucose cytes. Hexokinase was partially purified by a modification of 6-phosphate at physiological concentrations. Without inhibi- the method of Rijksen and Staal (9). Three hundred millili- tors or in the presence ofglucose 1,6-bisphosphate, 2,3-diphos- ters of blood was freed of leukocytes and platelets by pass- phoglycerate, and ADP, maximum hexokinase activity was ob- ing through a cellulose column (10), and the erythrocytes served at 5 mM glucose concentration. On the contrary, in the were washed 3 times with isotonic saline. The packed eryth- presence of glucose 6-phosphate, hexokinase activity increased rocytes were lysed with 1 vol of 0.4% saponin in H20 for 1 at glucose concentrations >5 mM; inhibition by glucose 6- hr. The hemolysate was mixed with 0.5 vol of 501% suspen- phosphate was partially competitive with glucose. The reliefby sion of DEAE-cellulose (DE52) equilibrated with 10 mM po- glucose of glucose 6-phosphate inhibition of hexokinase is a tassium phosphate buffer (pH 7.3) containing 3 mM 2-mer- possible explanation of the increased glucose 6-phosphate level captoethanol, and 3 mM NaF. After washing with the same in diabetic erythrocytes. buffer, the enzyme was eluted with 500 mM potassium phosphate buffer (pH 7.3) containing 3 mM 2-mercaptoethanol, 3 The erythrocytes of diabetic patients contain increased lev- mM NaF, and 10% (NH4)2SO4. Solid ammonium sulfate was els of the glycohemoglobin hemoglobin Al. The concentra- added to the enzyme solution to 75% saturation. After stand- tion of glucose 6-phosphate has been found to be increased ing overnight at 4TC the precipitate was collected by centrifu- in the erythrocytes of diabetic patients (1-3), and it has been gation, was dissolved in 10 mM potassium phosphate buffer suggested that glucose 6-phosphate may be the precursor of (pH 7.3) containing 3 mM 2-mercaptoethanol and 3 mM the carbohydrate variety of this glycohemoglobin. The NaF, and was dialyzed against the same buffer. The dialyzed mechanism by which erythrocyte glucose 6-phosphate levels solution was applied to a DEAE-cellulose column (0.8 x 23 become elevated in diabetics is by no means obvious, how- cm) equilibrated with the same buffer. The column was ever. The human erythrocyte is highly permeable to glucose. washed with the same buffer and then with the same buffer Human erythrocyte hexokinase has a very low Km for glu- containing 10 mM KCN. Elution of the enzyme was per- cose (50-80 x 10-6 M), and it is saturated with glucose even formed with a 500-ml linear gradient of0-500 mM KCI in the at normal plasma glucose levels (5 mM). Accordingly, the same buffer containing 10 mM KCN. Fractions of 5.5 ml increase in glucose 6-phosphate concentration in diabetic were collected and assayed for hexokinase, glucose-6-phos- erythrocytes is obviously not merely the direct result of in- phate dehydrogenase, glucose-phosphate isomerase, and creased availability of glucose to hexokinase. adenylate kinase (11). The fractions containing hexokinase It has been proposed that acidemia due to ketoacidosis activity and free ofglucose-6phosphate dehydrogenase, glu- sometimes seen in severe or untreated diabetics might cause cose phosphate isomerase, and adenylate kinase were elevation of glucose 6-phosphate levels in the erythrocytes pooled and concentrated by ultrafiltration. The enzyme was of diabetics, because phosphofructokinase activity is inhibit- stored at -20TC in 10 mM potassium phosphate buffer (pH ed by the lower pH (4). However, most diabetics are not 7.0) containing 3 mM 2-mercaptoethanol, 3 mM NaF, 2 mM acidotic. Moreover, Stevens et al. (1) and Tegos and Beutler glucose, and 10%6 (vol/vol) glycerol. Before use, the stored (3) observed no crossover in the levels of erythrocyte glyco- enzyme was dialyzed against 10 mM Tris HC1 (pH 8.0). lytic intermediates in diabetics. This suggested that high glu- Hexokinase Assay. The standard assay mixture (11) con- cose concentrations affected the hexokinase reaction itself. tained 50 mM Tris HCI, pH 8.0/0.2 mM NADP/1 mM Hexokinase normally operates in a state of partial inhibi- ATP/5 mM MgCl2/0.1 international unit (IU) of glucose-6- tion by some glycolytic intermediates (5-8). Relief of inhibi- phosphate dehydrogenase and glucose at the concentrations tion of hexokinase by high concentrations of glucose would indicated in the text. Absorbance was measured at 340 nm at result in the apparent activation of the enzyme by high glu- 37°C for 10-20 min. When glucose 6-phosvhate was studied as an inhibitor, it The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: IU, international unit(s). in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

1552 Downloaded by guest on September 28, 2021 Medical Sciences: Fujii and Beutler Proc. NatL Acad. Sci. USA 82 (1985) 1553 was necessary to measure hexokinase activity in another Studies with Purified Erythrocyte Hexokinase. Fig. 1 shows system in which ADP production was determined in the py- the effect of high concentrations of glucose on the inhibition ruvate kinase reaction. This assay system contained 50 mM of hexokinase by glucose 6-phosphate using the PK-LDH as- Tris HCl, pH 8.0/5 mM MgCl2/75 mM KCl/1 mM ATP/2 say method. Without glucose 6-phosphate, hexokinase activ- mM phosphoenolpyruvate/0.2 mM NADH/3 IU ofpyruvate ity was maximal at 5 mM glucose and 87.6% ± 1.5% (mean kinase/3 IU of lactate dehydrogenase. The reaction was fol- ± SEM) of the Vma at 0.5 mM glucose; glucose concentra- lowed by measuring optical density at 340 nm at 370C for 3 tions >5 mM resulted in no increase of activity. Since the min. One unit of enzyme activity was defined as the amount Km for glucose is 50-80 x 10-6 M, these findings were in of enzyme that catalyzes the formation of 1 umol of glucose perfect agreement with the predicted value from the Michae- 6-phosphate or 1 ,umol of ADP per min. In both systems, the lis-Menten equation, indicating the accuracy of the method reaction was started by adding 0.004 unit of hexokinase per used here. It is clear that hexokinase is completely saturated ml ofreaction mixture. A cuvette without hexokinase served at 5 mM glucose. In the presence of glucose 6-phosphate, on as the blank. the contrary, the activity is slightly, but significantly increased at glucose concentrations >5 mM. These results in- RESULTS dicate that hexokinase inhibition by glucose 6-phosphate is partially relieved by high concentrations of glucose. The Glucose 6-Phosphate Level After Incubation of Human data have been replotted in the inserted figure to provide Erythrocytes with Glucose. Fasting blood was drawn into insight into the relationship between glucose concentration heparin from normal volunteers. After centrifugation at 2000 and inhibition by glucose 6-phosphate. The curves obtained x g for 10 min, packed erythrocytes were suspended in 1 vol suggest the presence of a mixed type of inhibition (12). of 0.1 M Hepes buffer (pH 7.4) containing 135 mM NaCl in Using the standard assay, hexokinase activity in the ab- the presence of 5 mM and 30 mM glucose. Incubation was sence and presence of glucose 1,6-bisphosphate, 2,3-diphos- continued for 1 hr at 37°C in a shaking bath. The pH of both phoglycerate, and ADP at various glucose concentrations suspensions was the same at the end of the incubation. The was examined (data not given). In the absence of inhibitors, reaction was terminated by adding 4 vol of ice-cold 4% per- hexokinase activity reached its maximum activity at 5 mM chloric acid. The concentration of glucose 6-phosphate was glucose; higher glucose concentration resulted in no increase measured as described (11). In eight separate experiments, of activity. In the presence of physiological concentrations the glucose 6-phosphate concentration in the presence of 5 of glucose 1,6-bisphosphate (200 ,uM), 2,3-diphosphoglycer- mM glucose was found to be 30.19 ± 0.88 ,umol per liter of ate (5 mM), and ADP (1 mM), hexokinase activity also pla- packed erythrocytes (mean ± SEM); in the presence of 30 teaued at 5 mM and higher glucose concentrations, indicat- mM glucose, the glucose 6-phosphate concentration was ing that a high concentration of glucose is not competitive 35.8 ± 1.58 ,uM. The paired differences were 5.61 ± 1.79 ,uM with the action of these inhibitors. These findings are con- (mean ± SEM), this difference being statistically significant sistent with previous reports (7, 8). At higher concentrations atP < 0.05. of inhibitors, the same results were obtained.

110

u w 20 AM G6P 0 I- u , -: --- 0 M .6L - 30 ,uM G6P W) E 100 --- - .1 . - I- No G6P

- ._u 0.03- ._CZ 0ct - I I 0-0-0-o-.-.-o- NoG6P 90 - x c v 0.02-

IA)n ..AV M JP °0010 o- - 30j.gmG6 -- - 0.01 - - 30 ,uM G6P T 0.005 v/S 5 20 40 100 Glucose, mM FIG. 1. Effect of high glucose concentration on hexokinase inhibition by glucose 6-phosphate. Hexokinase activity is expressed as percent of the activity at 5 mM glucose at each concentration of glucose 6-phosphate. The mean values (± SEM) were obtained from several indepen- dent experiments and each experiment was performed in duplicate. (Inset) Eadie plot of the data. Initial velocity (v) is expressed as AA340/ min and glucose concentration, S, is in mM. G6P, glucose 6-phosphate. Downloaded by guest on September 28, 2021 1554 Medical Sciences: Fujii and Beutler Proc. NatL Acad Sci. USA 82 (1985) DISCUSSION phate dehydrogenase reaction depends primarily on the availability ofNADP and, hence, is not expected to be great- The elevated glucose 6-phosphate level that has been found ly affected by an increase in the glucose 6-phosphate level of in the erythrocytes of diabetics can also be documented in the erythrocytes. However, at physiological substrate con- normal erythrocytes that are incubated in vitro with high centrations, phosphofructokinase activity is strongly depen- concentrations of glucose under conditions of carefully con- dent on the level of fructose 6-phosphate, which, through trolled pH. Thus, it cannot be due to any ofthe variables that glucose phosphate isomerase, exists in a 1:3 equilibrium are incidental to the diabetic state. If the accumulation of mixture with glucose 6-phosphate in erythrocytes. Thus, the glucose 6-phosphate were due to inhibition of glucose phos- rate of removal of glucose 6-phosphate would be dependent phate isomerase, phosphofructokinase, or other glycolytic on its rate of formation and a new steady state would be enzymes, a decrease in metabolic intermediates downstream expected at glucose 6-phosphate levels that are roughly de- from the inhibited step would be expected, but no such pendent on the rate of glucose 6-phosphate formation by crossover has been detected in vivo (1, 3). Thus, it seemed hexokinase. The increase in hexokinase activity when the most likely that enhanced phosphorylation of glucose was glucose concentration was increased from 5 to 40 mM at a 30 responsible for the increased level of glucose 6-phosphate pM concentration of glucose 6-phosphate was 5.5%. By that occurred at high glucose concentrations. Since hexoki- comparison, the increase in the glucose 6-phosphate level nase was fully saturated with glucose even at a concentration found in intact erythrocyte incubated at 30 mM glucose of 5 mM, and since the enzyme normally functions under when compared with 5 mM glucose was 18.6%. Thus, the circumstances when it is under marked inhibition, relief of increases in hexokinase activity and of glucose 6-phosphate inhibition by glucose seemed a likely answer to the problem. levels were found to be of a similar order of magnitude. To our knowledge, however, no effect of high glucose con- These considerations indicate that our findings are consist- centration on the inhibitory kinetics of the enzyme had been ent with reported previously. Using the standard assay system, we the mechanism that we propose to account for the found the inhibitory effect of glucose 1,6-bisphosphate, 2,3- increased level of glucose 6-phosphate in erythrocytes ex- diphosphoglycerate, and ADP unaffected by glucose. The posed to high glucose concentrations. effect of glucose on inhibition by glucose 6-phosphate was This work was supported in part by National Institutes of Health more difficult to study, because it is this product of the reac- Division of Heart, Lung and Blood Grant HL25552 and Division of tion that is measured in the standard assay system. An assay Research Resources Grant RR00833. This is publication number was used in which the formation of ADP was estimated. In 3247BCR from the Research Institute of Scripps Clinic. using this approach, the accuracy of measurement of initial reaction velocity was of critical importance because during 1. Stevens, V. J., Vlassara, H., Abati, A. & Cerami, A. (1977) J. the reaction, glucose 6-phosphate accumulates rapidly, mak- Biol. Chem. 252, 2998-3002. ing it difficult to obtain a linear reaction rate. Only 0.004 2. McDonald, M. J., Shapiro, R., Bleichman, M., Solway, J. & ml was used and the reaction was Bunn, H. F. (1978) J. Biol. Chem. 253, 2327-2332. unit of hexokinase per 3. Tegos, C. & Beutler, E. (1980) 1. Lab. Clin. Med. 96, 85-89. recorded for only 3 min. These modifications and using 4. Kono, N., Kuwajima, M. & Tarui, S. (1981) Diabetes 30, 346- mean values obtained from several experiments made it pos- 353. sible to obtain reliable results. 5. Brewer, G. J. (1969) Biochim. Biophys. Acta 192, 157-161. The partial relief of hexokinase inhibition by glucose 6- 6. Beutler, E. (1971) Nature (London) New Biol. 232, 20-21. phosphate shown in our present work, although small, is 7. Gerber, G., Preissler, H., Heinrich, R. & Rapoport, S. M. probably sufficient to account for the modest accumulation (1974) Eur. J. Biochem. 45, 39-52. of glucose 6-phosphate in the erythrocytes of diabetic pa- 8. Rijksen, G. & Staal, G. E. J. (1977) Biochim. Biophys. Acta tients. The exact steady-state concentration of glucose 6- 485, 75-86. be be- 9. Rijksen, G. & Staal, G. E. J. (1976) Biochim. Biophys. Acta phosphate in erythrocytes cannot readily computed, 445, 330-341. cause it is a function not only ofhexokinase activity, but also 10. Beutler, E., West, C. & Blume, K. G. (1976) J. Lab. Clin. of the activities of phosphofructokinase and probably, to a Med. 88, 328-333. limited extent, of glucose-6-phosphate dehydrogenase. 11. Beutler, E. (1975) Red Cell Metabolism: A Manual of Bio- These activities, in turn, are controlled not only by their sub- chemical Methods (Grune & Stratton, New York), 2nd Ed. strates, but also by effectors including 2,3-diphosphoglycer- 12. Dixon, M. & Webb, E. C. (1964) Enzymes (Academic, Nev ate, ATP, ADP, NADP, and NADPH. The glucose-6-phos- York), 2nd Ed. 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