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Anomeric specificity of d -glucose metabolism in human erythrocytes
223
Clinica Chimica Acta, 172 (1988) 223-232 Elsevier
CCA 04090
Anomeric specificity of D-glucose metabolism in human erythrocytes F. Malaisse-Lagae, I. Sterling, A. Sener and W.J. Malaisse Laboratory of Experimental (Received
Medicine, Brussels Free University, Brussels (Belgium)
4 June 1987; revision received 8 October Key words: Erythrocytes;
D-Glucose
1987; accepted metabolism;
after revision
Anomeric
5 November
1987)
specificity
Summary The metabolism of D-[5-3H]glucose and D-[U-‘4C]glucose displays anomeric specificity in human erythrocytes. At close-to-physiological hexose concentrations, pure /_%D-[5-3H]glucose is metabolized at a higher rate than the corresponding pure c-Y-anomer in most, but not all, subjects. This situation represents a mirror image of that found in rat erythrocytes. The anomeric preference is modulated by the extracellular concentration of D-glucose and may differ for distinct metabolic variables. The anomeric specificity of D-glucose metabolism remains operative, whether at low or normal temperature, even in erythrocytes exposed to equilibrated D-glucose.
Introduction Although the anomeric specificity of enzymes catalyzing the early steps of hexose metabolism is known since several decades [l], the anomeric specificity of D-glucose (or D-mannose) metabolism in intact cells was only documented recently in rat pancreatic islets [2,3], erythrocytes [4], adipocytes [5] and hepatocytes [6]. The present study indicates that D-glucose metabolism displays anomeric specificity in human erythrocytes. Materials and methods Unlabelled (Y- and P-D-glucose were purchased from Sigma (St Louis, MO, USA). The labelled anomers of D-[5-3H]glucose and D-[U-‘4C]glucose were pre-
Correspondence to: W.J. Ma&se, M.D., Waterloo, B-1000 Brussels, Belgium.
0009-8981/88/$03.50
Laboratoire
0 1988 Elsevier Science Publishers
de Medecine
Experimentale,
B.V. (Biomedical
Division)
115, Boulevard
de
224
pared from solutions of labelled D-glucose in anomeric equilibrium (New England Nuclear, Boston, MA, USA) by a method described elsewhere [7]. D-[U-‘~C]G~Ucase, rather than D-[l-‘4C]glucose, was used for measuring both 14COz production and glucose 6-phosphate content in order to avoid isotopic dilution through recirculation in the pentose phosphate pathway. Antecubital venous blood (2 to 3 ml) was collected in heparinized syringes from 13 healthy individuals, aged 25 to 55, all members of our laboratory staff, and from 2 insulin-treated diabetic patients, aged 35 and 67. The erythrocytes were separated from the plasma and buffy coat, washed and resuspended in an iced glucose-free Krebs phosphate buffer by a procedure previously described [4]. The volume of erythrocytes in the reconstituted cell suspension amounted to either 20% (3H,0 production) or 40% (14COZ production). All results are expressed per ~1 of whole bood, assuming a hematocrit value of 40%. Aliquots of the cell suspension (20 ~1 for 3H,0 production and 120 ~1 for 14COZ production) were mixed with an equal volume of the same iced Krebs phosphate buffer containing the freshly dissolved anomers of D-glucose. After either 6 min incubation at 37” C or 60 min incubation at 9” C, the amount of ‘H,O [8] and 14C0, [9] formed by the erythrocytes was measured as described. The results were corrected for blank values found in the absence of cell and, in the case of D-[5-3H]glucose utilization, for the recovery of 3H,0. Two types of experiment were performed. In the first type, the metabolism of pure (Y- and /SD-glucose was compared, each medium containing only one of these anomers. In each experiment, the suspension of ‘erythrocytes prepared from a single blood sample was divided in 20 to 30 portions, so that 10 to 15 determinations could be made with each anomer. In the second type of experiments, 32 portions of the same suspension of erythrocytes were used, in groups of 8 samples each and under 4 distinct conditions, namely in media containing pure labelled a-D-glucose, pure labelled /?-D-glucose, a mixture of 36.2% labelled a-D-glucose and 63.8% unlabelled /3-D-glucose, or a mixture of 36.2% unlabelled a-D-glucose and 63.8% labelled P-D-glucose. In both types of experiment, some individuals were examined on 2 to 4 separate occasions. The 4 healthy individuals in whom the anomeric specificity of metabolism was examined at anomeric equilibrium are identified in this report by the letters A to D. In one series of experiments, the glucose 6-phosphate content of the erythrocytes was measured after 30 min incubation at 9” C in the presence of equilibrated D-[U;i4C]glucose. After centrifugation and removal of the incubation medium, the cell pellet (derived from 100 ~1 whole blood) was mixed with 250 ~1 perchloric acid (2.5%, w/v) containing carrier glucose 6-phosphate (150 pmol/l). After heating for 10 min at 60” C and centrifugation, an aliquot (200 ~1) of the supernatant medium was mixed with 100 ~1 of a Tris solution (0.2 mol/l) containing KOH (1.0 mol/l) and again centrifuged. Portions (100 ~1 each) of the neutralized extract were then mixed with an equal volume of a Tris/HCl buffer (100 mmol/l; pH 7.8) containing 10 mmol/l MgCl,, 50 mmol/l ammonium acetate, 2 mmol/l EDTA, 5 mmol/l NaHCO,, 1.0 mmol/l dithiothreitol, 1.0 mmol/l NADP+ and both yeast glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and yeast 6-phosphogluconate dehydro-
225
genase (EC 1.1.1.44) (each 0.6 U/ml; Boehringer, Mannheim, FRG). The tubes containing this mixture were placed in counting vials, which already contained 0.5 ml hyamine hydroxide (Packard, Downer Groves, IL, USA). After 60 min incubation at 37 o C, the reaction was halted by injecting 0.1 ml HCl (0.1 mol/l), and the i4C02 formed was recovered in the hyamine over a further 60 min incubation at 37” C. The results were corrected for the blank values found in the absence of enzyme and for the readings obtained with standard amounts of D-[U-i4C]glucose 6-phosphate prepared in extracts of unlabelled erythrocytes. All results are expressed as the mean (_t SEM) together with the number of individual measurements (n). The SEM on the sum or ratio between mean values was calculated as described elsewhere [lO,ll]. The statistical significance of differences was assessed by use of Student’s t test. In relating metabolic variables to the concentration of each anomer, their relative abundance in equilibrated solutions was taken as 36.2% and 63.8% for (Y- and P-D-glucose, respectively [12].
Results
-‘H,O production at 3 7 oC When incubated for 6 rnin at 37” C in the presence of a low concentration of D-glucose (0.1 mmol/l), the erythrocytes of subject A produced comparable amounts of 3H,0 (p > 0.4) from pure (Y- or /3-D-glucose (Table I). At anomeric equilibrium, the total production of 3H,0 was not significantly different from that found with pure P-D-[5-3H]glucose ( p = O.OS), but the relative contribution of a-D-glucose exceeded that expected from its relative abundance ( p < 0.02). A somewhat different situation was encountered when the concentration of D-glucose was raised to 7.0 mmol/l. Indeed, the /3-anomer was now more efficiently metabolized than the a-anomer (p < 0.001). At anomeric equilibrium, the total production of 3H,0 remained not significantly different from that found with pure /&D-[5-3H]glucose (p > 0.2), but the relative contribution of each anomer was now no more different from their respective abundance ( p > 0.5).
TABLE I Utilization
of D-[5-3H]ghrcose
at 37 o C by erythrocytes 0.1
D-glucose (mmol/l)
‘H,O production
(pmol/pl Pure ~-D-glucose Pure /3-D-glucose Equilibrated D-glucose
Relative contribution a-D-glucose P-D-ghlcose
blood per
at anomeric
of subject
A 7.0
6 min) 99.1 f 2.5 (16) 96.5 f 2.7 (16) 89.5 f 2.1 (16)
99.8 k4.5 (16) 134.8 + 2.4 (16) 130.7k2.1 (16)
equilibrium (%) 39.7* 1.3 (16) 60.3 f 2.0 (16)
35.8kO.6 (16) 64.2 f 1.3 (16)
226 TABLE
II
Utilization D-Glucose
of D-[5-3H]gIucose
at 9 o C by erythrocytes
(mmol/l)
0.1
‘Ha0 production (pmol/pl Pure a-D-glucose Pure P-D-glucose Equilibrated D-glucose Relative contribution a-D-ghicose
blood per 60 min) 70.4 + 2.0 (32) 71.2+1.4(32) 67.9 + 1.2 (32)
at anomeric
P-D-ghCOSe
equilibrium (9) 42.Ok 1.0 (32) 58.1 5 1.2 (32)
of subject
A
1.0
7.0
129.2 f 4.5 (24) 135.8 & 2.4 (24) 141.5 + 4.0 (24)
138.3 + 3.4 (24) 145.5 f 2.3 (24) 148.2 f 2.3 (24)
39.6 f 2.1 (24) 60.4 k 2.0 (24)
38.2 * 0.9 (24) 61.8 _t 1.2 (24)
“H,O production at 9 ‘C
Further experiments were performed over 60 n-tin incubation at 9’ C. When the erythrocytes of subject A were incubated at low concentrations of D-glucose (0.1 or 1.0 mmol/l), no anomeric difference in the production of 3H,0 was observed in the presence of the pure anomers. At a higher level of D-glucose (7.0 mmol/l), however, the P-anomer was more efficiently metabolized than the a-anomer (p < 0.05). At anomeric equilibrium, the total production of 3H,0 was not significantly different from that found with pure fi-D-[5-3H]glucose, whatever the concentration of the hexose ( p > 0.05 or more). The relative contribution of a-D-glucose clearly exceeded that expected from its relative abundance at the lowest concentration (0.1 mmol/l) of the hexose (p < O.OOl), but such a difference was less marked at higher concentrations of D-glucose (1.0 and 7.0 mmol/l), in which case it either failed to achieve statistical significance or yielded a probability just below 0.05. The comparison of the data listed in Tables I and II indicates that a rise in D-glucose concentration exerted comparable effects upon the anomeric specificity of D-[53H]glucose utilization whether at 9 or 37’ C. 14C02 production at 9°C
In the last series of experiments performed with erythrocytes of subject A, the production of i4C02 from D-[U-‘4C]glucose was measured over 60 min incubation at 9 o C in the presence of 0.1 mmol/l D-glucose (Table III). Unexpectedly, a-D-&case was more efficiently oxidized than P-D-glucose ( p < 0.005). At anomeric equilibrium, the total production of 14COz was close to that found with pure a-D-glucose, and the relative contribution of the a-anomer largely exceeded its relative abundance ( p < 0.005). ‘H20 production in other subjects
In subject A, the utilization of pure a-D-[5-3H]glucose (0.1 mmol/l) at 9” C did not exceed that of &D-[5-3H]glucose (Table II). In three other subjects (B to D), however, the mean value for 3H,0 production from a-D-[5-3H]glucose, used at the same low concentration (0.1 mmol/l), exceeded (p < 0.005) the mean value found with P-D-[5-3H]glucose (Table Iv>. At anomeric equilibrium, the total production of
227 TABLE
III
Oxidation
of
D-Glucose
(mmol/l)
at 9’ C by erythrocytes
D-[U-14C]ghCOSe
of subject
A
0.1
i4C02 production (fmol/pI Pure a-D-glucose Pure P-D-glucose Equilibrated D-glucose Relative contribution
blood per 60 min) 413.6k25.3 (24) 318.1+ 18.0 (24) 410.0 * 23.5 (24)
at anomeric
equilibrium
(S)
a-D-glucose
50.0 i 4.2 (24) 50.0 k 3.8 (24)
P-D-glucose
3H,0
was not significantly different from that found with pure P-D-[S3H]glucose but the relative contribution of the a-anomer again exceeded that expected from its relative abundance (p < 0.005). The difference in the anomeric specificity of D-glucose metabolism in subjects A and B to D, respectively, does not represent an exceptional feature. Thus, in a series
( p > 0.2)
1.1 l-
,;
l*O____
-_
_-_
____
--
‘, 1 i ;
0.9-
0
I
I” 0 C ._
0.8-
.o % K
0.7-
-0 $
1
I
..
. .
II
ii I
I
i . .
Tl
1
-
. . .. 11
.. . . I1
. II”“”
Fig. 1. The CX//S ratios for the utilization of the pure anomers of D-glucose by erythrocytes of 11 normal (0) and 2 diabetic (0) individuals, as measured over 60 min incubation at 9 o C in the presence of 4.0 mmol/l D-[5-3H]glucose, are ranged in order of increasing values. The SEM on the individual ratios is derived from 9 to 15 measurements made with each anomer in each experiment. Certain individuals were examined on two (mh, ma, mu) or four 6s) separate occasions. The absolute value for /3-D-[5-3H]ghIcose utilization in the healthy individuals averaged 137X* 3.0 pmol/nl blood per 60 min (n =163). Also shown is the statistical significance of differences from unity ( *p < 0.05; * *p < 0.005). The column to the right indicates the mean value ( + SEM) derived from the 13 individual mean ratios.
228 TABLE
IV
Utilization
of D-[5-3H]glucose
D-Glucose
(mmol/l)
3H,0
production
PUrC
at 9 o C by erythrocytes
(pmol/pl
blood per 60 min) 60.7 f 1.7 (24) 52.3 +2.0 (24) 55.0 + 1.3 (24)
Pure P-D-glucose Equilibrated
B to D
0.1
a-D-ghCOSe
D-glucose
Relative contribution
of subjects
at anomeric
equilibrium
(a) 42.2 f 1.7 (24) 57.8 * 2.1 (24)
a-D-ghCOSC P-D-glucose
of measurements made in 13 distinct persons (11 healthy subjects and 2 diabetics), the a//3 ratio in glucose utilization, measured over 60 min incubation at 9 o C in the presence of 4.0 mmol/l D-[5-3H]glucose, varied from 0.760 to 1.025 (Fig. 1). In 8 of these 13 subjects, the a//? ratio was significantly lower than unity. The overall mean value for the LX//~ ratio amounted to 0.899 f 0.022 (n = 13; p < 0.001 as compared to unity). It should be underlined that all these measurements were performed in the presence of 4.0 mmol/l D-glucose, at which concentration the utilization of the hexose is close to its maximal value (Fig. 2). The glucose
t
0.1
D-glucose
(m M)
Fig. 2. Effect of increasing concentrations of D-glucose upon the production of 3H,0 from equilibrated D-[5-3H]ghtcose by erythrocytes obtained from 3 healthy individuals (mh, mu, js) and incubated for 60 mm at 9 o C. Mean values (k SEM) are derived from 38 to 97 individual measurements performed in a series of 5 separate experiments.
229 6-phosphate content of the erythrocytes, measured after 30 min incubation at 9°C increased from 13.2 k 1.0 pmol/pl blood in the presence of 0.1 mmol/l D-glucose to 17.2 k 1.0 and 19.6 1- 1.7 pmol/pl in the presence of 1.0 and 7.0 mmol/l D-glucose, respectively (n = 9 to 20). From the latter data and those illustrated in Fig. 2, it can be calculated that the fractional turnover rate of the pool of endogenous glucose 6-phosphate does not exceed O.l/min, whatever the extracellular concentration of the hexose. Relationship between the metabolism of pure anomers and their relative contribution to the metabolism of equilibrated D-glucose Figure 3 illustrates the positive correlation (r = 0.8584; n = 7, p < 0.05) between the relative contribution of the P-anomer to the metabolism of equilibrated D-glucose and the corresponding /?/a ratio for the metabolism of the pure anomers. The mean results illustrated in this figure are taken from Tables I-IV and were thus obtained at two temperatures (9 and 37OC), at increasing concentrations of D-glucose (0.1, 1.0 and 7.0 mmol/l), in erythrocytes removed from distinct subjects, and for two distinct metabolic parameters (3H,0 and i4COz production). Despite such a heterogeneity in experimental conditions, there appeared to exist a single relationship between the two variables under consideration. Discussion The present results afford five new pieces of information. First, they reveal that the metabolism of D-glucose displays anomeric specificity in human erythrocytes. The numerous factors possibly responsible for such a specificity were recently examined in the case of D-[U-‘4C]glucose oxidation by human erythrocytes [13]. Second, the data illustrated in Fig. 1 indicate that, at a. close-to-physiological glucose concentration, the utilization of D-[5-3H]glucose by human erythrocytes is, as a rule, higher with pure /3- than a-D-glucose. This represents a mirror image of that found with rat erythrocytes [4,14] and, hence, supports the concept that the anomeric specificity of hexose metabolism underwent a phylogenetic evolution [15,16]. Third, our work suggests that the relative contribution of the (Y- and p-anomer to the overall catabolism of equilibrated D-glucose in a given metabolic pathway is modulated by the efficiency of each anomer, considered separately, to be metabolized in that pathway (Fig. 3). However, under suitable conditions, the relative contribution of a-D-glucose to the metabolism of equilibrated D-glucose exceeded that expected from its relative abundance, although no anomeric difference was found in the metabolism of the pure anomers (Table II, first column). The most likely explanation for such a situation resides in both the greater affinity of hexokinase for a- than /3-D-glucose [13,17] and the relatively low fractional turnover rates of the glucose 6-phosphate pool in human erythrocytes. Fourth, our work provides the first demonstration that the anomeric specificity of D-glucose metabolism may be operative, even at 37” C, in cells exposed to
230
+ 8
1 I , I 1
22 0 0
0.7
1 0.0
0.9
p/a
1.0
ratio
1.1
(pure
1.2
1.3
1.4
anomers)
Fig. 3. Relationship between the relative contribution of /I-D-glucose to the overall metabolism of of pure anomers. Mean equilibrated r@ucose and the corresponding p/ cx ratio for the metabolism values ( f SEM) are taken from Tables I to IV. The dotted vertical line refers to the situation in which no difference is observed in the metabolism of pure anomers. The dotted horizontal line refers to the situation in which the relative contribution of /3-D-ghCOSe to the overall metabolism of equilibrated D-glucose is proportional to the abundancy of the /3-anomer (ie 0.638).
equilibrated D-glucose. This supports the view that the anomeric specificity of enzymes participates in the regulation of D-glucose metabolism under physiological conditions [ 151. Last, the present data indicate that, even under standardized conditions, the anomeric specificity of D-glucose metabolism in human erythrocytes displays significant individual variations. Further work is required to investigate whether such variations could be used as a finger-print when considering individual differences in the regulation of anomeric processes such as the secretion of insulin by the pancreatic B-cell [18].
Acknowledgements
This work was supported by grants from the Belgian Foundation for Scientific Medical Research. I.S. is a Research Fellow of the Belgian National Foundation for Scientific Research. The authors thank S.P. Dufrane, M. Mahy, J. Schoonheydt, M. Urbain and C. Demesmaeker for technical assistance.
231
References 1 Benkovic SJ, Schray KJ. The anomeric specificity of glycolytic enzymes. Adv Enzymol 1976;44:139-164. 2 Malaisse WJ, Sener A, Levy J. The stimulus-secretion coupling of glucose-induced insulin release. XXIV. Metabolism of a- and P-D-ghCOSC in isolated islets. J Biol Chem 1976;251:5936-5943. 3 Sener A, Malaisse-Lagae F, Lebrun P, Herchuelz A, Leclercq-Meyer V, Malaisse WJ. Anomeric specificity of D-mannose metabolism in pancreatic islets. Biochem Biophys Res Commun 1982;108:1567-1573. 4 Malaisse WJ, Giroix M-H, Dufrane SP, MaIaisse-Lagae F, Sener A. Anomeric specificity of glycolysis in a non glucokinase-containing cell. Biochem Int 1985;10:233-240. 5 Malaisse-Lagae F, Malaisse WJ. Anomeric specificity of n-glucose metabolism in rat adipocytes. Eur J Biochem 1986;158:663-666. 6 Malaisse WJ. Anomeric specificity of n-glucose utilization in rat hepatocytes. IRCS Med Sci 1986;14:609-610. 7 Sener A, Leclercq-Meyer V, Marchand J, Giroix M-H, Dufrane SP, Malaisse WJ. Is glucokinase responsible for the anomeric specificity of glycolysis in pancreatic islets? J Biol Chem 195;260:12978-12981. 8 Levy J, Herchuelz A, Sener A, Malaisse-Lagae F, Malaisse W. Cytochalasin B-induced impairment of glucose metabolism in islets of Langerhans. Endocrinology 1976;98:429-437. 9 Carpinelli AR, Sener A, Herchuelz A, Malaisse WJ. Stimulus-secretion coupling of glucose-induced insulin release. Effect of intracellular acidification upon calcium efflux from islet cells. Metab Clin Exp 1980;29:540-545. 10 Sener A, Malaisse-Lagae F, Malaisse WJ. The stimulus-secretion coupling of glucose-induced insulin release. Environmental influences on L-glutamine oxidation in pancreatic islets. Biochem J 1982;202:309-316. 11 Sener A, Malaisse-Lagae F, Dufrane SP, Malaisse WJ. The coupling of metabolic to secretory events in pancreatic islets. The cytosolic redox state. Biochem J 1984;200:433-440. 12 Wurster B, Hess B. The reaction of hexokinase with equilibrated D-ghcose. Em J Biochem 1973;36:68-71. 13 Malaisse-Lagae F, Malaisse WJ. Anomeric specificity of D-glucose phosphorylation and oxidation in’ human erythrocytes. Int J Biochem 1987;19:733-736. 14 Ma&se WJ, Giroix M-H, Dufrane SP, Malaisse-Lagae F, Sener A. Environmental modulation of the anomeric specificity of glucose metabolism, in normal and tumoral cells. Biochim Biophys Acta 1985;847:48-52. 15 Malaisse WJ, Malaisse-Lagae F, Sener A. Anomericspecificity of hexose metabolism in pancreatic islets. Physiol Rev 1983;63:773-786. 16 Malaisse-Lagae F, Giroix M-H, Sener A, Ma&se WJ. Temperature dependency of the anomeric specificity of yeast and bovine hexokinases. Biol Chem Hoppe-Seyler 1986;367:411-416. 17 Giroix M-H, Sener A, Malaisse WJ. Reciprocal influence of glucose anomers upon their respective phosphorylation by hexokinase. Biol Chem Hoppe-Seyler 1986;367:47-51. 18 Rovira A, Garrote FJ, Valverde I, MaIaisse WJ. Anomeric specificity of glucose-induced insulin release in normal and diabetic subjects. Diabetes Res 1987;5:119-124.
Clinica Chimica Acta, 172 (1988) 223-232 Elsevier
CCA 04090
Anomeric specificity of D-glucose metabolism in human erythrocytes F. Malaisse-Lagae, I. Sterling, A. Sener and W.J. Malaisse Laboratory of Experimental (Received
Medicine, Brussels Free University, Brussels (Belgium)
4 June 1987; revision received 8 October Key words: Erythrocytes;
D-Glucose
1987; accepted metabolism;
after revision
Anomeric
5 November
1987)
specificity
Summary The metabolism of D-[5-3H]glucose and D-[U-‘4C]glucose displays anomeric specificity in human erythrocytes. At close-to-physiological hexose concentrations, pure /_%D-[5-3H]glucose is metabolized at a higher rate than the corresponding pure c-Y-anomer in most, but not all, subjects. This situation represents a mirror image of that found in rat erythrocytes. The anomeric preference is modulated by the extracellular concentration of D-glucose and may differ for distinct metabolic variables. The anomeric specificity of D-glucose metabolism remains operative, whether at low or normal temperature, even in erythrocytes exposed to equilibrated D-glucose.
Introduction Although the anomeric specificity of enzymes catalyzing the early steps of hexose metabolism is known since several decades [l], the anomeric specificity of D-glucose (or D-mannose) metabolism in intact cells was only documented recently in rat pancreatic islets [2,3], erythrocytes [4], adipocytes [5] and hepatocytes [6]. The present study indicates that D-glucose metabolism displays anomeric specificity in human erythrocytes. Materials and methods Unlabelled (Y- and P-D-glucose were purchased from Sigma (St Louis, MO, USA). The labelled anomers of D-[5-3H]glucose and D-[U-‘4C]glucose were pre-
Correspondence to: W.J. Ma&se, M.D., Waterloo, B-1000 Brussels, Belgium.
0009-8981/88/$03.50
Laboratoire
0 1988 Elsevier Science Publishers
de Medecine
Experimentale,
B.V. (Biomedical
Division)
115, Boulevard
de
224
pared from solutions of labelled D-glucose in anomeric equilibrium (New England Nuclear, Boston, MA, USA) by a method described elsewhere [7]. D-[U-‘~C]G~Ucase, rather than D-[l-‘4C]glucose, was used for measuring both 14COz production and glucose 6-phosphate content in order to avoid isotopic dilution through recirculation in the pentose phosphate pathway. Antecubital venous blood (2 to 3 ml) was collected in heparinized syringes from 13 healthy individuals, aged 25 to 55, all members of our laboratory staff, and from 2 insulin-treated diabetic patients, aged 35 and 67. The erythrocytes were separated from the plasma and buffy coat, washed and resuspended in an iced glucose-free Krebs phosphate buffer by a procedure previously described [4]. The volume of erythrocytes in the reconstituted cell suspension amounted to either 20% (3H,0 production) or 40% (14COZ production). All results are expressed per ~1 of whole bood, assuming a hematocrit value of 40%. Aliquots of the cell suspension (20 ~1 for 3H,0 production and 120 ~1 for 14COZ production) were mixed with an equal volume of the same iced Krebs phosphate buffer containing the freshly dissolved anomers of D-glucose. After either 6 min incubation at 37” C or 60 min incubation at 9” C, the amount of ‘H,O [8] and 14C0, [9] formed by the erythrocytes was measured as described. The results were corrected for blank values found in the absence of cell and, in the case of D-[5-3H]glucose utilization, for the recovery of 3H,0. Two types of experiment were performed. In the first type, the metabolism of pure (Y- and /SD-glucose was compared, each medium containing only one of these anomers. In each experiment, the suspension of ‘erythrocytes prepared from a single blood sample was divided in 20 to 30 portions, so that 10 to 15 determinations could be made with each anomer. In the second type of experiments, 32 portions of the same suspension of erythrocytes were used, in groups of 8 samples each and under 4 distinct conditions, namely in media containing pure labelled a-D-glucose, pure labelled /?-D-glucose, a mixture of 36.2% labelled a-D-glucose and 63.8% unlabelled /3-D-glucose, or a mixture of 36.2% unlabelled a-D-glucose and 63.8% labelled P-D-glucose. In both types of experiment, some individuals were examined on 2 to 4 separate occasions. The 4 healthy individuals in whom the anomeric specificity of metabolism was examined at anomeric equilibrium are identified in this report by the letters A to D. In one series of experiments, the glucose 6-phosphate content of the erythrocytes was measured after 30 min incubation at 9” C in the presence of equilibrated D-[U;i4C]glucose. After centrifugation and removal of the incubation medium, the cell pellet (derived from 100 ~1 whole blood) was mixed with 250 ~1 perchloric acid (2.5%, w/v) containing carrier glucose 6-phosphate (150 pmol/l). After heating for 10 min at 60” C and centrifugation, an aliquot (200 ~1) of the supernatant medium was mixed with 100 ~1 of a Tris solution (0.2 mol/l) containing KOH (1.0 mol/l) and again centrifuged. Portions (100 ~1 each) of the neutralized extract were then mixed with an equal volume of a Tris/HCl buffer (100 mmol/l; pH 7.8) containing 10 mmol/l MgCl,, 50 mmol/l ammonium acetate, 2 mmol/l EDTA, 5 mmol/l NaHCO,, 1.0 mmol/l dithiothreitol, 1.0 mmol/l NADP+ and both yeast glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and yeast 6-phosphogluconate dehydro-
225
genase (EC 1.1.1.44) (each 0.6 U/ml; Boehringer, Mannheim, FRG). The tubes containing this mixture were placed in counting vials, which already contained 0.5 ml hyamine hydroxide (Packard, Downer Groves, IL, USA). After 60 min incubation at 37 o C, the reaction was halted by injecting 0.1 ml HCl (0.1 mol/l), and the i4C02 formed was recovered in the hyamine over a further 60 min incubation at 37” C. The results were corrected for the blank values found in the absence of enzyme and for the readings obtained with standard amounts of D-[U-i4C]glucose 6-phosphate prepared in extracts of unlabelled erythrocytes. All results are expressed as the mean (_t SEM) together with the number of individual measurements (n). The SEM on the sum or ratio between mean values was calculated as described elsewhere [lO,ll]. The statistical significance of differences was assessed by use of Student’s t test. In relating metabolic variables to the concentration of each anomer, their relative abundance in equilibrated solutions was taken as 36.2% and 63.8% for (Y- and P-D-glucose, respectively [12].
Results
-‘H,O production at 3 7 oC When incubated for 6 rnin at 37” C in the presence of a low concentration of D-glucose (0.1 mmol/l), the erythrocytes of subject A produced comparable amounts of 3H,0 (p > 0.4) from pure (Y- or /3-D-glucose (Table I). At anomeric equilibrium, the total production of 3H,0 was not significantly different from that found with pure P-D-[5-3H]glucose ( p = O.OS), but the relative contribution of a-D-glucose exceeded that expected from its relative abundance ( p < 0.02). A somewhat different situation was encountered when the concentration of D-glucose was raised to 7.0 mmol/l. Indeed, the /3-anomer was now more efficiently metabolized than the a-anomer (p < 0.001). At anomeric equilibrium, the total production of 3H,0 remained not significantly different from that found with pure /&D-[5-3H]glucose (p > 0.2), but the relative contribution of each anomer was now no more different from their respective abundance ( p > 0.5).
TABLE I Utilization
of D-[5-3H]ghrcose
at 37 o C by erythrocytes 0.1
D-glucose (mmol/l)
‘H,O production
(pmol/pl Pure ~-D-glucose Pure /3-D-glucose Equilibrated D-glucose
Relative contribution a-D-glucose P-D-ghlcose
blood per
at anomeric
of subject
A 7.0
6 min) 99.1 f 2.5 (16) 96.5 f 2.7 (16) 89.5 f 2.1 (16)
99.8 k4.5 (16) 134.8 + 2.4 (16) 130.7k2.1 (16)
equilibrium (%) 39.7* 1.3 (16) 60.3 f 2.0 (16)
35.8kO.6 (16) 64.2 f 1.3 (16)
226 TABLE
II
Utilization D-Glucose
of D-[5-3H]gIucose
at 9 o C by erythrocytes
(mmol/l)
0.1
‘Ha0 production (pmol/pl Pure a-D-glucose Pure P-D-glucose Equilibrated D-glucose Relative contribution a-D-ghicose
blood per 60 min) 70.4 + 2.0 (32) 71.2+1.4(32) 67.9 + 1.2 (32)
at anomeric
P-D-ghCOSe
equilibrium (9) 42.Ok 1.0 (32) 58.1 5 1.2 (32)
of subject
A
1.0
7.0
129.2 f 4.5 (24) 135.8 & 2.4 (24) 141.5 + 4.0 (24)
138.3 + 3.4 (24) 145.5 f 2.3 (24) 148.2 f 2.3 (24)
39.6 f 2.1 (24) 60.4 k 2.0 (24)
38.2 * 0.9 (24) 61.8 _t 1.2 (24)
“H,O production at 9 ‘C
Further experiments were performed over 60 n-tin incubation at 9’ C. When the erythrocytes of subject A were incubated at low concentrations of D-glucose (0.1 or 1.0 mmol/l), no anomeric difference in the production of 3H,0 was observed in the presence of the pure anomers. At a higher level of D-glucose (7.0 mmol/l), however, the P-anomer was more efficiently metabolized than the a-anomer (p < 0.05). At anomeric equilibrium, the total production of 3H,0 was not significantly different from that found with pure fi-D-[5-3H]glucose, whatever the concentration of the hexose ( p > 0.05 or more). The relative contribution of a-D-glucose clearly exceeded that expected from its relative abundance at the lowest concentration (0.1 mmol/l) of the hexose (p < O.OOl), but such a difference was less marked at higher concentrations of D-glucose (1.0 and 7.0 mmol/l), in which case it either failed to achieve statistical significance or yielded a probability just below 0.05. The comparison of the data listed in Tables I and II indicates that a rise in D-glucose concentration exerted comparable effects upon the anomeric specificity of D-[53H]glucose utilization whether at 9 or 37’ C. 14C02 production at 9°C
In the last series of experiments performed with erythrocytes of subject A, the production of i4C02 from D-[U-‘4C]glucose was measured over 60 min incubation at 9 o C in the presence of 0.1 mmol/l D-glucose (Table III). Unexpectedly, a-D-&case was more efficiently oxidized than P-D-glucose ( p < 0.005). At anomeric equilibrium, the total production of 14COz was close to that found with pure a-D-glucose, and the relative contribution of the a-anomer largely exceeded its relative abundance ( p < 0.005). ‘H20 production in other subjects
In subject A, the utilization of pure a-D-[5-3H]glucose (0.1 mmol/l) at 9” C did not exceed that of &D-[5-3H]glucose (Table II). In three other subjects (B to D), however, the mean value for 3H,0 production from a-D-[5-3H]glucose, used at the same low concentration (0.1 mmol/l), exceeded (p < 0.005) the mean value found with P-D-[5-3H]glucose (Table Iv>. At anomeric equilibrium, the total production of
227 TABLE
III
Oxidation
of
D-Glucose
(mmol/l)
at 9’ C by erythrocytes
D-[U-14C]ghCOSe
of subject
A
0.1
i4C02 production (fmol/pI Pure a-D-glucose Pure P-D-glucose Equilibrated D-glucose Relative contribution
blood per 60 min) 413.6k25.3 (24) 318.1+ 18.0 (24) 410.0 * 23.5 (24)
at anomeric
equilibrium
(S)
a-D-glucose
50.0 i 4.2 (24) 50.0 k 3.8 (24)
P-D-glucose
3H,0
was not significantly different from that found with pure P-D-[S3H]glucose but the relative contribution of the a-anomer again exceeded that expected from its relative abundance (p < 0.005). The difference in the anomeric specificity of D-glucose metabolism in subjects A and B to D, respectively, does not represent an exceptional feature. Thus, in a series
( p > 0.2)
1.1 l-
,;
l*O____
-_
_-_
____
--
‘, 1 i ;
0.9-
0
I
I” 0 C ._
0.8-
.o % K
0.7-
-0 $
1
I
..
. .
II
ii I
I
i . .
Tl
1
-
. . .. 11
.. . . I1
. II”“”
Fig. 1. The CX//S ratios for the utilization of the pure anomers of D-glucose by erythrocytes of 11 normal (0) and 2 diabetic (0) individuals, as measured over 60 min incubation at 9 o C in the presence of 4.0 mmol/l D-[5-3H]glucose, are ranged in order of increasing values. The SEM on the individual ratios is derived from 9 to 15 measurements made with each anomer in each experiment. Certain individuals were examined on two (mh, ma, mu) or four 6s) separate occasions. The absolute value for /3-D-[5-3H]ghIcose utilization in the healthy individuals averaged 137X* 3.0 pmol/nl blood per 60 min (n =163). Also shown is the statistical significance of differences from unity ( *p < 0.05; * *p < 0.005). The column to the right indicates the mean value ( + SEM) derived from the 13 individual mean ratios.
228 TABLE
IV
Utilization
of D-[5-3H]glucose
D-Glucose
(mmol/l)
3H,0
production
PUrC
at 9 o C by erythrocytes
(pmol/pl
blood per 60 min) 60.7 f 1.7 (24) 52.3 +2.0 (24) 55.0 + 1.3 (24)
Pure P-D-glucose Equilibrated
B to D
0.1
a-D-ghCOSe
D-glucose
Relative contribution
of subjects
at anomeric
equilibrium
(a) 42.2 f 1.7 (24) 57.8 * 2.1 (24)
a-D-ghCOSC P-D-glucose
of measurements made in 13 distinct persons (11 healthy subjects and 2 diabetics), the a//3 ratio in glucose utilization, measured over 60 min incubation at 9 o C in the presence of 4.0 mmol/l D-[5-3H]glucose, varied from 0.760 to 1.025 (Fig. 1). In 8 of these 13 subjects, the a//? ratio was significantly lower than unity. The overall mean value for the LX//~ ratio amounted to 0.899 f 0.022 (n = 13; p < 0.001 as compared to unity). It should be underlined that all these measurements were performed in the presence of 4.0 mmol/l D-glucose, at which concentration the utilization of the hexose is close to its maximal value (Fig. 2). The glucose
t
0.1
D-glucose
(m M)
Fig. 2. Effect of increasing concentrations of D-glucose upon the production of 3H,0 from equilibrated D-[5-3H]ghtcose by erythrocytes obtained from 3 healthy individuals (mh, mu, js) and incubated for 60 mm at 9 o C. Mean values (k SEM) are derived from 38 to 97 individual measurements performed in a series of 5 separate experiments.
229 6-phosphate content of the erythrocytes, measured after 30 min incubation at 9°C increased from 13.2 k 1.0 pmol/pl blood in the presence of 0.1 mmol/l D-glucose to 17.2 k 1.0 and 19.6 1- 1.7 pmol/pl in the presence of 1.0 and 7.0 mmol/l D-glucose, respectively (n = 9 to 20). From the latter data and those illustrated in Fig. 2, it can be calculated that the fractional turnover rate of the pool of endogenous glucose 6-phosphate does not exceed O.l/min, whatever the extracellular concentration of the hexose. Relationship between the metabolism of pure anomers and their relative contribution to the metabolism of equilibrated D-glucose Figure 3 illustrates the positive correlation (r = 0.8584; n = 7, p < 0.05) between the relative contribution of the P-anomer to the metabolism of equilibrated D-glucose and the corresponding /?/a ratio for the metabolism of the pure anomers. The mean results illustrated in this figure are taken from Tables I-IV and were thus obtained at two temperatures (9 and 37OC), at increasing concentrations of D-glucose (0.1, 1.0 and 7.0 mmol/l), in erythrocytes removed from distinct subjects, and for two distinct metabolic parameters (3H,0 and i4COz production). Despite such a heterogeneity in experimental conditions, there appeared to exist a single relationship between the two variables under consideration. Discussion The present results afford five new pieces of information. First, they reveal that the metabolism of D-glucose displays anomeric specificity in human erythrocytes. The numerous factors possibly responsible for such a specificity were recently examined in the case of D-[U-‘4C]glucose oxidation by human erythrocytes [13]. Second, the data illustrated in Fig. 1 indicate that, at a. close-to-physiological glucose concentration, the utilization of D-[5-3H]glucose by human erythrocytes is, as a rule, higher with pure /3- than a-D-glucose. This represents a mirror image of that found with rat erythrocytes [4,14] and, hence, supports the concept that the anomeric specificity of hexose metabolism underwent a phylogenetic evolution [15,16]. Third, our work suggests that the relative contribution of the (Y- and p-anomer to the overall catabolism of equilibrated D-glucose in a given metabolic pathway is modulated by the efficiency of each anomer, considered separately, to be metabolized in that pathway (Fig. 3). However, under suitable conditions, the relative contribution of a-D-glucose to the metabolism of equilibrated D-glucose exceeded that expected from its relative abundance, although no anomeric difference was found in the metabolism of the pure anomers (Table II, first column). The most likely explanation for such a situation resides in both the greater affinity of hexokinase for a- than /3-D-glucose [13,17] and the relatively low fractional turnover rates of the glucose 6-phosphate pool in human erythrocytes. Fourth, our work provides the first demonstration that the anomeric specificity of D-glucose metabolism may be operative, even at 37” C, in cells exposed to
230
+ 8
1 I , I 1
22 0 0
0.7
1 0.0
0.9
p/a
1.0
ratio
1.1
(pure
1.2
1.3
1.4
anomers)
Fig. 3. Relationship between the relative contribution of /I-D-glucose to the overall metabolism of of pure anomers. Mean equilibrated r@ucose and the corresponding p/ cx ratio for the metabolism values ( f SEM) are taken from Tables I to IV. The dotted vertical line refers to the situation in which no difference is observed in the metabolism of pure anomers. The dotted horizontal line refers to the situation in which the relative contribution of /3-D-ghCOSe to the overall metabolism of equilibrated D-glucose is proportional to the abundancy of the /3-anomer (ie 0.638).
equilibrated D-glucose. This supports the view that the anomeric specificity of enzymes participates in the regulation of D-glucose metabolism under physiological conditions [ 151. Last, the present data indicate that, even under standardized conditions, the anomeric specificity of D-glucose metabolism in human erythrocytes displays significant individual variations. Further work is required to investigate whether such variations could be used as a finger-print when considering individual differences in the regulation of anomeric processes such as the secretion of insulin by the pancreatic B-cell [18].
Acknowledgements
This work was supported by grants from the Belgian Foundation for Scientific Medical Research. I.S. is a Research Fellow of the Belgian National Foundation for Scientific Research. The authors thank S.P. Dufrane, M. Mahy, J. Schoonheydt, M. Urbain and C. Demesmaeker for technical assistance.
231
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