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Alpha- and beta-anomeric preference of glucose-induced insulin secretion at physiological and higher glucose concentrations, respectively
Vol. 180, No. 2, 1991 October 31, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 709-715
ALPHA- AND BETA-ANOMERIC PREFERENCE OF GLUCOSE-INDUCED INSULIN SECRETION AT PHYSIOLOGICAL AND HIGHER GLUCOSE CONCENTRATIONS, RESPECTIVELY lchitomo Miwa, Tomiyasu Murata, and Jun Okuda Department of Clinical Biochemistry, Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya 468, Japan Received September 9, 1991
We determined the anomeric preference of glucose phosphorylation by islet glucokinase, glucose utilization by pancreatic islets, and insulin secretion induced by glucose over a wide range of glucose concentrations. a-D-Glucose was phosphorylated faster than 13-D-glucose by islet glucokinase at lower glucose concentrations (5 and 10 mM), whereas the opposite anomeric preference was observed at higher glucose concentrations (40 and 60 mM). At 20 mM, there was no significant difference in phosphorylation rate between the two anomers. Similar patterns of anomeric preference were observed both in islet glucose utilization and in glucose-induced insulin secretion. The present study affords strong evidence that glucokinase is responsible for the anomeric preference of glucose-stimulated insulin secretion through anomeric discrimination in islet glucose utilization. ©1991AcadomicP..... ~nc.
It is widely accepted that pancreatic islets discriminate between the ct and 13 anomers of glucose over the physiological range of glucose concentrations (below 15 mM) with respect to stimulation of insulin secretion (1-5). The ct-anomeric preference in glucose-induced insulin release was initially considered as supportive evidence for the presence of a glucoreceptor that recognizes glucose as a signal for insulin release at the plasma membrane of pancreatic B-cells (1, 2). Malaisse et al., however, suggested that the higher insulinotropic capacity of the ct anomer of glucose, as distinct from the 13 anomer, might be explained by ~t-preferential glucose utilization due to the stereospecificity of the enzymes glucose-6-phosphate isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 5.4.2.2) for the tx anomer of glucose 6-phosphate (6, 7). A newer explanation is that the preference of glucokinase (EC 2.7.1.1) for the a anomer of glucose at physiological concentrations of the hexose leads to a-preferential metabolism and insulinotropism of glucose
709
0006-291X/91 $1.50 Copyright 6) 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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(8, 9). There is, however, no convincing evidence for any of these three views. With these conflicting views in mind, our attention was paid to a report that glucokinases from liver and insulinoma of rats phosphorylate a-D-glucose more rapidly than I~-D-glucose at glucose concentrations below 15 mM and that the anomeric preference is reversed at glucose concentrations above 20 mM (8). If the third view is valid and islet glucokinase shows a similar anomeric preference, it would be expected that both glucose utilization and glucose-induced insulin secretion would also be preferential for the 0t anomer and the f~ anomer of glucose at lower and higher glucose concentrations, respectively. The aim of this study was to examine the validity of the third view. MATERIALS AND METHODS Pancreatic islets were isolated from fed male Sprague-Dawley rats (300-350 g, Clea Japan, Tokyo) as described previously (10). Rat islet glucokinase was prepared from pancreatic islet supernatant by chromatography on DEAE Affi-Gel Blue (Bio-Rad, Richmond, CA) according to the method of Meglasson et al. (11) except that the pH of the buffer was 7.0. The enzyme was eluted in a single peak, whereas Meglasson et al. reported the occurrence of a major peak and a minor one. Phosphorylation of glucose anomers by glucokinase was determined by fluorometry according to the method of Meglasson and Matschinsky (8) except that reactions were carried out for 3 min at 37 °C. The anomeric purity of a- and 13-D-glucose (Pfanstiehl, Waukegan, IL) was determined enzymatically as described previously (12) and was found to be greater than 98 % for both anomers. A solution of D-[5-3H]glucose (10,900 Ci/mol) was purchased from New England Nuclear (Boston, MA). Alpha- and beta-anomers (5.1 Ci/mol) of D-[5-3H]glucose were prepared as described previously (13). The anomeric purity of the preparation~ as determined by our enzymatic method (12), was 92 % for a-D-[5-H]glucose and 96 % for ~-D-[5-3H]glucose. Batches of 30 islets were preincubated for 30 min at 37 °C in plastic microtubes (capacity, 1.5 ml) that contained 200 ~tl of buffer A (118 mM NaCI, 4.7 mM KCI, 2.5 mM CaCI2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 24.2 mM NaHCO3, 0.2 % bovine serum albumin, and 2.8 mM glucose [pH 7.4]). Buffer A was used after equilibration with 5% CO2/95% 02. After removal of this buffer, the islets were incubated for 3 min at 37 °C in 80 ~tl of buffer A containing a- or ~-D-[5-3H]glucose of indicated concentrations instead of 2.8 mM glucose. Media alone (80 ~tl each) as blanks were also incubated in parallel. The incubation media were prepared by dissolving each anomer of D-[5-3H]glucose in ice-cold glucose-free buffer A just before use. The NaCI concentration of buffer A was decreased to 95 mM to avoid too high osmolarity of the incubation medium in experiments with 60 mM glucose anomers. After incubation, 10 ~tl of 3 M HC1 and 40 ~tl of ethanol were added to the microtubes to stop metabolism. After mixing, a piece of filter paper (13 x 30 mm, No.2, Toyo Roshi, Tokyo) was put into each microtube so that almost all of solution was absorbed in an attempt to facilitate the diffusion of ~H20. The microtubes were then placed in 20-ml glass scintillation vials that contained 0.5 ml of distilled water. The vials were stoppered and ke~t at 30 °C for 24 hr for accomplishment of diffusion of 3H20. Eighty rtl of H20 standard (2 x 109 dpm/1, New England Nuclear, Boston, MA) was treated 710
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in the same way to correct for incomplete equilibration during the diffusion step. Radioactivity that diffused from the microtubes into 0.5 ml of water was determined. Recovery of the 3H20 standard was 95.3 _+ 3.3 % (mean _+ SD, n=9) of the theoretical value. The rate of glucose utilization was calculated by the formula described by Ashcroft et al. (14). For measurement of insulin secretion from rat pancreatic islets, batches of 5 islets were placed in plastic cylinders (0.7 cm ID x 4.5 cm) covered with nylon mesh of 106-~tm pore size at one end. The islets were preincubated for 30 min at 37 °C in 1.0 ml of buffer A. Preincubation and subsequent incubation were performed by placement of each of the plastic cylinders in fiat-bottomed vials (1.4 cm ID x 4.2 cm). The islets were incubated in 0.5 ml of buffer A containing a- or 13-D-glucose of indicated concentrations instead of 2.8 mM glucose. Basal insulin release was determined by incubation of islets in 0.5 ml of buffer A alone. In experiments with 60 mM a- and 13-D-glucose, the NaC1 concentration of buffer A was changed to 95 mM. Stock solutions of each anomer of various concentrations (10 times the final concentrations) were prepared with ice-cold glucose-free buffer A just before use. Incubation was initiated by addition of 50 vl of stock solution. To minimize the detrimental influence of anomerization of glucose anomers on the discrimination between a-D-glucose- and 13-D-glucose-stimulated insulin secretion, we performed three consecutive incubations (2 min each), with changing of the incubation medium between, for each batch of 5 islets. The three incubation media (0.5 ml each) were pooled, and insulin was measured by an enzyme immunoassay with a kit from Mitsui (Tokyo). Statistical differences between groups were evaluated by Student's unpaired t-test. The ZZtest was performed to assess the significance of differences between percentages. The criterion of significance was set at P<0.05. RESULTS Alpha-D-glucose was phosphorylated faster than 13-D-glucose by islet glucokinase at lower glucose concentrations (5 and 10 mM), whereas the opposite anomeric preference was observed at higher glucose concentrations (40 and 60 mM) (Fig.l). At 20 mM, there was no significant anomeric preference. This result is similar to those results reported for rat liver and insulinoma glucokinases (8). If glucokinase regulates glycolysis in islets, glucose usage must show an anomeric preference similar to that found in the glucose phosphorylation by glucokinase. Indeed, the anomeric preference of glucose usage determined by 3H20 formation from a- and [3-D-[5)H]glucose looked like a reflection of that of glucose phosphorylation over a wide range of glucose concentrations (up to 60 mM) (Fig.2). With respect to glucose-induced insulin secretion, the well-known a-anomeric preference at physiological glucose concentrations (below 15 mM) (1-5) was confirmed in the present study (Fig.3). In addition, we found that the fl anomer of glucose is a better stimulant than the a anomer at higher glucose concentrations (40 and 60 mM). 711
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
150.
12--~ 10 ~
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CO
>, o
8'
E
cL
o_ 50.
0 Q
0
5
10
20
40
60
0 0
k&)
Glucose ( mM )
5
10
20
40
Glucose ( mM )
Fig. 1. Phosphorylation of a- and 13-D-glucose by glucokinase prepared from rat pancreatic islets. Glucokinase was incubated for 3 min at 37 °C in the presence of a-D-gluocse (O) or 13-D-glucose (O). Data are expressed as the mean percentage _+ SD of the phosphorylation rate (1.92 nmol/3 min) observed with 20 mM a-D-glucose; n=5. *P<0.001 vs. ~-D-glucose. Fig. 2. Utilization of a-D-[5)H]glucose and I~-D-[5)H]glucose in rat pancreatic islets. Isolated pancreatic islets were incubated for 3 min at 37 °C in the presence of a-D-[5)H]glucose ( e ) or 13-D-[5-3H]glucose (O), and the radioactivity of 3H20 formed was measured. Data represent the mean _+ SEM of 7 experiments. *P<0.01, **P<0.001 vs. 13-D-glucose.
120
1 O0
.=_ E £o
E
12
9
40
60
80 ¸ 6o
12
6
10
20
4O i 03 ._=
0 i
Glucose ( mM )
Fig. 3. Insulin secretion from rat pancreatic islets exposed to either a-D-glucose or I~-D-glucose. Isolated pancreatic islets were incubated three times consecutively (2 min each) at 37 °C, with changing the incubation medium between, in the presence of a-D-glucose ( O ) or ~-D-glucose ( O ) , and insulin released was measured by enzyme immunoassay. Each value is expressed by subtraction of the basal insulin release (54 fmol/6 min/islet) from the observed insulin release. Data represent the mean _+ SEM of the number of experiments indicated near each symbol. *P<0.001 vs. 13-D-glucose. 712
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DISCUSSION Malaisse et al. (15) reported that rat pancreatic islets produced 3H20 more rapidly from a-D-[5-3H]glucose than from 13-D-[5-3H]glucose (va/v~3 =1.7) at a glucose concentration of 5.6 mM when incubation was carried out for 60 min at 7 °C. They also described, however, that the a and anomers of D-[5-3H]glucose did not differ in their rate of conversion to 3H20 at a glucose concentration of 40 mM under the same incubation conditions (16). Idahl et al. (17) reported that there was no significant difference in glycolysis by microdissected pancreatic islets of ob/ob mice between a- and [3-D-[5-3H]glucose (over a concentration range of 3 to 21 mM) when the islets were incubated for 3 min at 37 °C. Discrepancies between these and our results may be due to differences in the method of incubation, the origin of islets, and/or the radioisotopic purity of a- and 13-D-[5-3H]glucose. There is only one study that examined the anomeric preference of glucose-stimulated insulin secretion at such a high glucose concentration as 40 mM (16), and no anomeric preference was observed in that study. Isolated pancreatic islets were employed in our study, whereas isolated perfused pancreata, in which the phenomenon of B-cell memory (18) is an inevitable problem, were used in the other study. This difference may be a possible cause for the discrepancy in anomeric preference between the two studies. Beta-D-glucose 6-phosphate formed from 13-D-glucose by glucokinase is changed to a-D-glucose 6-phosphate by spontaneous and catalytic (by glucose-6-phosphate isomerase) reactions (19) and then isomerized to fructose 6-phosphate by glucose-6-phosphate isomerase, which acts specifically on the a anomer of glucose 6-phosphate (19, 20). The isomerization of glucose 6-phosphate to fructose 6-phosphate, thus, is likely to be one of the rate-limiting steps in glycolysis when [3-D-glucose 6-phosphate is produced at a high rate. It appears that this is a reason for less distinct differences between the a and [3 anomers of glucose in glucose utilization relative to glucose phosphorylation at high glucose concentrations (40 and 60 mM). The extent of anomeric preference in glucose utilization was also different from that of glucose-induced insulin secretion. It has been reported that glucose-induced insulin secretion, as a function of islet glucose utilization, shows a typical sigmoidal curve (21). This sigmoidicity can be considered as the cause of the discrepancy in the extent of anomeric preference between the two parameters at physiological glucose concentrations. At the present time, however, we do not have any appropriate explanation for the more marked anomeric preference noted 713
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in insulin secretion than in glucose utilization at glucose concentrations exceeding 20 mM. Phosphorylation of glucose in pancreatic islets is not solely catalyzed by glucokinase. Hexokinase also phosphorylates glucose in islets. However, this enzyme is severely inhibited by hexose phosphates such as glucose 6-phosphate and glucose 1,6-bisphosphate in intact islets (22). In addition, phosphorylation of glucose by hexokinase can not account for the concentration dependency of glucose utilization in pancreatic islets by any means, since hexokinase activity will not vary with increased glucose concentration because of full saturation of the enzyme (Km for glucose = 0.05 mM) at glucose concentrations above 1 mM. At physiological and higher glucose concentrations, hence, an increase in islet glucose phosphorylation is thought to occur primarily by glucokinase. From the kinetic data of glucokinase with glucose anomers, it has been proposed that glucokinase may be implicated in the established greater potency of a-D-glucose at physiological concentrations as a stimulant of insulin secretion (8, 9). However, the view has not yet been evidenced. The present study strongly suggested that glucokinase is responsible for the anomeric preference of both glucose utilization and glucose-induced insulin secretion in pancreatic islets over a wide range of glucose concentrations. This is the first direct evidence for the view. REFERENCES l Niki, A., Niki, H., Miwa, I., and Okuda, J. (1974) Science 186, 150-151. 2. Grodsky, G. M., Fanska, R., West, L., and Manning, M. (1974) Science 186, 536-538. 3 Rossini, A. A., Soeldner, J. S., Hiebert, J. M., Weir, G. C., and Gleason, R. E. (1974) Diabetologia 10, 795-799. 4 Grodsky, G. M., Fanska, R., and Lundquist, I. (1975) Endocrinology 97, 573-580. 5 Matschinsky, F. M., Pagliara, A. S., Hover, B. A., Haymond, M. W., and Stillings, S. N. (1975) Diabetes 24, 369-372. 6 Malaisse, W. J., Sener, A., Koser, M., and Herchuelz, A. (1976) J. Biol. Chem. 251, 5936-5943. 7 Malaisse, W. J., Malaisse-Lagae, F., and Sener, A. (1983) Physiol. Rev. 63, 773-786. 8 Meglasson, M. D., and Matschinsky, F. M. (1983) J. Biol. Chem. 258, 6705 -6708. 9 Miwa, I., Inagaki, K., and Okuda, J. (1983) Biochem. Int. 7, 449-454. 10. Miwa, I., Murata, T., Mitsuyama, S., and Okuda, J. (1990) Diabetes 39, 1170-1176. 11. Meglasson, M. D., Burch, P. T., Berner, D. K., Najafi, H., Vogin, A. P., and Matschinsky, F. M. (1983) Proc. Natl. Acad. Sci. USA 80, 85-89. 12. Okuda, J., Miwa, I., Maeda, K., and Tokui, K. (1977) Carbohydr. Res. 58, 267-270. 13. Miwa, I., Okuda, J., Niki, H., and Niki, A. (1975) J. Biochem. 78, 1109-1111. .
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1 4. Ashcroft, S. J. H., Weerasinghe, L. C. C., Bassett, J. M., and Randle, P. J. (1972) Biochem. J. 126, 525-532. 15. Malaisse, W. J., Giroix, M.-H., and Sener, A. (1985) J. Biol. Chem. 260, 14630-14632. 16. Sener, A., Giroix, M.-H., Leclercq-Meyer, V., Marchand, J., and Malaisse, W. J. (1985) Biochem. Int. 11, 77-84. 17. Idahl, L.-A., Rahemtulla. F., Sehlin, J., and T~iljedal, I.-B. (1976) Diabetes 25, 450-458. 18. Grill, V., Adamson, U., and Cerasi, E. (1978) J. Clin. Invest. 61, 1034-1043. 19. Benkovic, S., and Schray, K. J. (1976) Adv. Enzymol. 44, 139-164. 20. Malaisse, W. J., Sener, A., Koser, M., and Herchuelz, A. (1976) FEBS Lett. 65, 131-134. 21. Meglasson, M. D., and Matschinsky, F. M. (1986) Diabetes Metab. Rev. 2, 163-214. 22. Giroix, M.-H., Sener, A., Pipeleers, D. G., and Malaisse, W. J. (1984) Biochem. J. 223, 447-453.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 709-715
ALPHA- AND BETA-ANOMERIC PREFERENCE OF GLUCOSE-INDUCED INSULIN SECRETION AT PHYSIOLOGICAL AND HIGHER GLUCOSE CONCENTRATIONS, RESPECTIVELY lchitomo Miwa, Tomiyasu Murata, and Jun Okuda Department of Clinical Biochemistry, Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya 468, Japan Received September 9, 1991
We determined the anomeric preference of glucose phosphorylation by islet glucokinase, glucose utilization by pancreatic islets, and insulin secretion induced by glucose over a wide range of glucose concentrations. a-D-Glucose was phosphorylated faster than 13-D-glucose by islet glucokinase at lower glucose concentrations (5 and 10 mM), whereas the opposite anomeric preference was observed at higher glucose concentrations (40 and 60 mM). At 20 mM, there was no significant difference in phosphorylation rate between the two anomers. Similar patterns of anomeric preference were observed both in islet glucose utilization and in glucose-induced insulin secretion. The present study affords strong evidence that glucokinase is responsible for the anomeric preference of glucose-stimulated insulin secretion through anomeric discrimination in islet glucose utilization. ©1991AcadomicP..... ~nc.
It is widely accepted that pancreatic islets discriminate between the ct and 13 anomers of glucose over the physiological range of glucose concentrations (below 15 mM) with respect to stimulation of insulin secretion (1-5). The ct-anomeric preference in glucose-induced insulin release was initially considered as supportive evidence for the presence of a glucoreceptor that recognizes glucose as a signal for insulin release at the plasma membrane of pancreatic B-cells (1, 2). Malaisse et al., however, suggested that the higher insulinotropic capacity of the ct anomer of glucose, as distinct from the 13 anomer, might be explained by ~t-preferential glucose utilization due to the stereospecificity of the enzymes glucose-6-phosphate isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 5.4.2.2) for the tx anomer of glucose 6-phosphate (6, 7). A newer explanation is that the preference of glucokinase (EC 2.7.1.1) for the a anomer of glucose at physiological concentrations of the hexose leads to a-preferential metabolism and insulinotropism of glucose
709
0006-291X/91 $1.50 Copyright 6) 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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(8, 9). There is, however, no convincing evidence for any of these three views. With these conflicting views in mind, our attention was paid to a report that glucokinases from liver and insulinoma of rats phosphorylate a-D-glucose more rapidly than I~-D-glucose at glucose concentrations below 15 mM and that the anomeric preference is reversed at glucose concentrations above 20 mM (8). If the third view is valid and islet glucokinase shows a similar anomeric preference, it would be expected that both glucose utilization and glucose-induced insulin secretion would also be preferential for the 0t anomer and the f~ anomer of glucose at lower and higher glucose concentrations, respectively. The aim of this study was to examine the validity of the third view. MATERIALS AND METHODS Pancreatic islets were isolated from fed male Sprague-Dawley rats (300-350 g, Clea Japan, Tokyo) as described previously (10). Rat islet glucokinase was prepared from pancreatic islet supernatant by chromatography on DEAE Affi-Gel Blue (Bio-Rad, Richmond, CA) according to the method of Meglasson et al. (11) except that the pH of the buffer was 7.0. The enzyme was eluted in a single peak, whereas Meglasson et al. reported the occurrence of a major peak and a minor one. Phosphorylation of glucose anomers by glucokinase was determined by fluorometry according to the method of Meglasson and Matschinsky (8) except that reactions were carried out for 3 min at 37 °C. The anomeric purity of a- and 13-D-glucose (Pfanstiehl, Waukegan, IL) was determined enzymatically as described previously (12) and was found to be greater than 98 % for both anomers. A solution of D-[5-3H]glucose (10,900 Ci/mol) was purchased from New England Nuclear (Boston, MA). Alpha- and beta-anomers (5.1 Ci/mol) of D-[5-3H]glucose were prepared as described previously (13). The anomeric purity of the preparation~ as determined by our enzymatic method (12), was 92 % for a-D-[5-H]glucose and 96 % for ~-D-[5-3H]glucose. Batches of 30 islets were preincubated for 30 min at 37 °C in plastic microtubes (capacity, 1.5 ml) that contained 200 ~tl of buffer A (118 mM NaCI, 4.7 mM KCI, 2.5 mM CaCI2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 24.2 mM NaHCO3, 0.2 % bovine serum albumin, and 2.8 mM glucose [pH 7.4]). Buffer A was used after equilibration with 5% CO2/95% 02. After removal of this buffer, the islets were incubated for 3 min at 37 °C in 80 ~tl of buffer A containing a- or ~-D-[5-3H]glucose of indicated concentrations instead of 2.8 mM glucose. Media alone (80 ~tl each) as blanks were also incubated in parallel. The incubation media were prepared by dissolving each anomer of D-[5-3H]glucose in ice-cold glucose-free buffer A just before use. The NaCI concentration of buffer A was decreased to 95 mM to avoid too high osmolarity of the incubation medium in experiments with 60 mM glucose anomers. After incubation, 10 ~tl of 3 M HC1 and 40 ~tl of ethanol were added to the microtubes to stop metabolism. After mixing, a piece of filter paper (13 x 30 mm, No.2, Toyo Roshi, Tokyo) was put into each microtube so that almost all of solution was absorbed in an attempt to facilitate the diffusion of ~H20. The microtubes were then placed in 20-ml glass scintillation vials that contained 0.5 ml of distilled water. The vials were stoppered and ke~t at 30 °C for 24 hr for accomplishment of diffusion of 3H20. Eighty rtl of H20 standard (2 x 109 dpm/1, New England Nuclear, Boston, MA) was treated 710
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in the same way to correct for incomplete equilibration during the diffusion step. Radioactivity that diffused from the microtubes into 0.5 ml of water was determined. Recovery of the 3H20 standard was 95.3 _+ 3.3 % (mean _+ SD, n=9) of the theoretical value. The rate of glucose utilization was calculated by the formula described by Ashcroft et al. (14). For measurement of insulin secretion from rat pancreatic islets, batches of 5 islets were placed in plastic cylinders (0.7 cm ID x 4.5 cm) covered with nylon mesh of 106-~tm pore size at one end. The islets were preincubated for 30 min at 37 °C in 1.0 ml of buffer A. Preincubation and subsequent incubation were performed by placement of each of the plastic cylinders in fiat-bottomed vials (1.4 cm ID x 4.2 cm). The islets were incubated in 0.5 ml of buffer A containing a- or 13-D-glucose of indicated concentrations instead of 2.8 mM glucose. Basal insulin release was determined by incubation of islets in 0.5 ml of buffer A alone. In experiments with 60 mM a- and 13-D-glucose, the NaC1 concentration of buffer A was changed to 95 mM. Stock solutions of each anomer of various concentrations (10 times the final concentrations) were prepared with ice-cold glucose-free buffer A just before use. Incubation was initiated by addition of 50 vl of stock solution. To minimize the detrimental influence of anomerization of glucose anomers on the discrimination between a-D-glucose- and 13-D-glucose-stimulated insulin secretion, we performed three consecutive incubations (2 min each), with changing of the incubation medium between, for each batch of 5 islets. The three incubation media (0.5 ml each) were pooled, and insulin was measured by an enzyme immunoassay with a kit from Mitsui (Tokyo). Statistical differences between groups were evaluated by Student's unpaired t-test. The ZZtest was performed to assess the significance of differences between percentages. The criterion of significance was set at P<0.05. RESULTS Alpha-D-glucose was phosphorylated faster than 13-D-glucose by islet glucokinase at lower glucose concentrations (5 and 10 mM), whereas the opposite anomeric preference was observed at higher glucose concentrations (40 and 60 mM) (Fig.l). At 20 mM, there was no significant anomeric preference. This result is similar to those results reported for rat liver and insulinoma glucokinases (8). If glucokinase regulates glycolysis in islets, glucose usage must show an anomeric preference similar to that found in the glucose phosphorylation by glucokinase. Indeed, the anomeric preference of glucose usage determined by 3H20 formation from a- and [3-D-[5)H]glucose looked like a reflection of that of glucose phosphorylation over a wide range of glucose concentrations (up to 60 mM) (Fig.2). With respect to glucose-induced insulin secretion, the well-known a-anomeric preference at physiological glucose concentrations (below 15 mM) (1-5) was confirmed in the present study (Fig.3). In addition, we found that the fl anomer of glucose is a better stimulant than the a anomer at higher glucose concentrations (40 and 60 mM). 711
Vol. 180, No. 2, 1991
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150.
12--~ 10 ~
f
E 100.
CO
>, o
8'
E
cL
o_ 50.
0 Q
0
5
10
20
40
60
0 0
k&)
Glucose ( mM )
5
10
20
40
Glucose ( mM )
Fig. 1. Phosphorylation of a- and 13-D-glucose by glucokinase prepared from rat pancreatic islets. Glucokinase was incubated for 3 min at 37 °C in the presence of a-D-gluocse (O) or 13-D-glucose (O). Data are expressed as the mean percentage _+ SD of the phosphorylation rate (1.92 nmol/3 min) observed with 20 mM a-D-glucose; n=5. *P<0.001 vs. ~-D-glucose. Fig. 2. Utilization of a-D-[5)H]glucose and I~-D-[5)H]glucose in rat pancreatic islets. Isolated pancreatic islets were incubated for 3 min at 37 °C in the presence of a-D-[5)H]glucose ( e ) or 13-D-[5-3H]glucose (O), and the radioactivity of 3H20 formed was measured. Data represent the mean _+ SEM of 7 experiments. *P<0.01, **P<0.001 vs. 13-D-glucose.
120
1 O0
.=_ E £o
E
12
9
40
60
80 ¸ 6o
12
6
10
20
4O i 03 ._=
0 i
Glucose ( mM )
Fig. 3. Insulin secretion from rat pancreatic islets exposed to either a-D-glucose or I~-D-glucose. Isolated pancreatic islets were incubated three times consecutively (2 min each) at 37 °C, with changing the incubation medium between, in the presence of a-D-glucose ( O ) or ~-D-glucose ( O ) , and insulin released was measured by enzyme immunoassay. Each value is expressed by subtraction of the basal insulin release (54 fmol/6 min/islet) from the observed insulin release. Data represent the mean _+ SEM of the number of experiments indicated near each symbol. *P<0.001 vs. 13-D-glucose. 712
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DISCUSSION Malaisse et al. (15) reported that rat pancreatic islets produced 3H20 more rapidly from a-D-[5-3H]glucose than from 13-D-[5-3H]glucose (va/v~3 =1.7) at a glucose concentration of 5.6 mM when incubation was carried out for 60 min at 7 °C. They also described, however, that the a and anomers of D-[5-3H]glucose did not differ in their rate of conversion to 3H20 at a glucose concentration of 40 mM under the same incubation conditions (16). Idahl et al. (17) reported that there was no significant difference in glycolysis by microdissected pancreatic islets of ob/ob mice between a- and [3-D-[5-3H]glucose (over a concentration range of 3 to 21 mM) when the islets were incubated for 3 min at 37 °C. Discrepancies between these and our results may be due to differences in the method of incubation, the origin of islets, and/or the radioisotopic purity of a- and 13-D-[5-3H]glucose. There is only one study that examined the anomeric preference of glucose-stimulated insulin secretion at such a high glucose concentration as 40 mM (16), and no anomeric preference was observed in that study. Isolated pancreatic islets were employed in our study, whereas isolated perfused pancreata, in which the phenomenon of B-cell memory (18) is an inevitable problem, were used in the other study. This difference may be a possible cause for the discrepancy in anomeric preference between the two studies. Beta-D-glucose 6-phosphate formed from 13-D-glucose by glucokinase is changed to a-D-glucose 6-phosphate by spontaneous and catalytic (by glucose-6-phosphate isomerase) reactions (19) and then isomerized to fructose 6-phosphate by glucose-6-phosphate isomerase, which acts specifically on the a anomer of glucose 6-phosphate (19, 20). The isomerization of glucose 6-phosphate to fructose 6-phosphate, thus, is likely to be one of the rate-limiting steps in glycolysis when [3-D-glucose 6-phosphate is produced at a high rate. It appears that this is a reason for less distinct differences between the a and [3 anomers of glucose in glucose utilization relative to glucose phosphorylation at high glucose concentrations (40 and 60 mM). The extent of anomeric preference in glucose utilization was also different from that of glucose-induced insulin secretion. It has been reported that glucose-induced insulin secretion, as a function of islet glucose utilization, shows a typical sigmoidal curve (21). This sigmoidicity can be considered as the cause of the discrepancy in the extent of anomeric preference between the two parameters at physiological glucose concentrations. At the present time, however, we do not have any appropriate explanation for the more marked anomeric preference noted 713
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in insulin secretion than in glucose utilization at glucose concentrations exceeding 20 mM. Phosphorylation of glucose in pancreatic islets is not solely catalyzed by glucokinase. Hexokinase also phosphorylates glucose in islets. However, this enzyme is severely inhibited by hexose phosphates such as glucose 6-phosphate and glucose 1,6-bisphosphate in intact islets (22). In addition, phosphorylation of glucose by hexokinase can not account for the concentration dependency of glucose utilization in pancreatic islets by any means, since hexokinase activity will not vary with increased glucose concentration because of full saturation of the enzyme (Km for glucose = 0.05 mM) at glucose concentrations above 1 mM. At physiological and higher glucose concentrations, hence, an increase in islet glucose phosphorylation is thought to occur primarily by glucokinase. From the kinetic data of glucokinase with glucose anomers, it has been proposed that glucokinase may be implicated in the established greater potency of a-D-glucose at physiological concentrations as a stimulant of insulin secretion (8, 9). However, the view has not yet been evidenced. The present study strongly suggested that glucokinase is responsible for the anomeric preference of both glucose utilization and glucose-induced insulin secretion in pancreatic islets over a wide range of glucose concentrations. This is the first direct evidence for the view. REFERENCES l Niki, A., Niki, H., Miwa, I., and Okuda, J. (1974) Science 186, 150-151. 2. Grodsky, G. M., Fanska, R., West, L., and Manning, M. (1974) Science 186, 536-538. 3 Rossini, A. A., Soeldner, J. S., Hiebert, J. M., Weir, G. C., and Gleason, R. E. (1974) Diabetologia 10, 795-799. 4 Grodsky, G. M., Fanska, R., and Lundquist, I. (1975) Endocrinology 97, 573-580. 5 Matschinsky, F. M., Pagliara, A. S., Hover, B. A., Haymond, M. W., and Stillings, S. N. (1975) Diabetes 24, 369-372. 6 Malaisse, W. J., Sener, A., Koser, M., and Herchuelz, A. (1976) J. Biol. Chem. 251, 5936-5943. 7 Malaisse, W. J., Malaisse-Lagae, F., and Sener, A. (1983) Physiol. Rev. 63, 773-786. 8 Meglasson, M. D., and Matschinsky, F. M. (1983) J. Biol. Chem. 258, 6705 -6708. 9 Miwa, I., Inagaki, K., and Okuda, J. (1983) Biochem. Int. 7, 449-454. 10. Miwa, I., Murata, T., Mitsuyama, S., and Okuda, J. (1990) Diabetes 39, 1170-1176. 11. Meglasson, M. D., Burch, P. T., Berner, D. K., Najafi, H., Vogin, A. P., and Matschinsky, F. M. (1983) Proc. Natl. Acad. Sci. USA 80, 85-89. 12. Okuda, J., Miwa, I., Maeda, K., and Tokui, K. (1977) Carbohydr. Res. 58, 267-270. 13. Miwa, I., Okuda, J., Niki, H., and Niki, A. (1975) J. Biochem. 78, 1109-1111. .
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1 4. Ashcroft, S. J. H., Weerasinghe, L. C. C., Bassett, J. M., and Randle, P. J. (1972) Biochem. J. 126, 525-532. 15. Malaisse, W. J., Giroix, M.-H., and Sener, A. (1985) J. Biol. Chem. 260, 14630-14632. 16. Sener, A., Giroix, M.-H., Leclercq-Meyer, V., Marchand, J., and Malaisse, W. J. (1985) Biochem. Int. 11, 77-84. 17. Idahl, L.-A., Rahemtulla. F., Sehlin, J., and T~iljedal, I.-B. (1976) Diabetes 25, 450-458. 18. Grill, V., Adamson, U., and Cerasi, E. (1978) J. Clin. Invest. 61, 1034-1043. 19. Benkovic, S., and Schray, K. J. (1976) Adv. Enzymol. 44, 139-164. 20. Malaisse, W. J., Sener, A., Koser, M., and Herchuelz, A. (1976) FEBS Lett. 65, 131-134. 21. Meglasson, M. D., and Matschinsky, F. M. (1986) Diabetes Metab. Rev. 2, 163-214. 22. Giroix, M.-H., Sener, A., Pipeleers, D. G., and Malaisse, W. J. (1984) Biochem. J. 223, 447-453.
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